Anti-tigit antibodies and methods of use

ABSTRACT

The invention provides anti-TIGIT (T-cell immunoreceptor with Ig and ITIM domains) antibodies and methods of using the same.

FIELD OF THE INVENTION

The present invention relates to anti-TIGIT (T-cell immunoreceptor withIg and ITIM domains) antibodies and methods of using the same.

BACKGROUND OF THE INVENTION

Immune-related diseases (e.g., cancer) are the manifestation orconsequence of complex biological pathways, which in normal physiologyare critical for responding to insult or injury, initiating repair frominsult or injury, and mounting innate and acquired defenses. Disease orpathology occurs when these normal physiological pathways causeadditional insult or injury that is directly related to the intensity ofthe response (e.g., as a consequence of abnormal regulation or excessivestimulation) or as a reaction to self.

Although the genesis of these diseases often involves multi-steppathways and often multiple different biological systems/pathways,intervention at critical points in one or more of these pathways canhave an ameliorative or therapeutic effect. Therapeutic intervention canoccur by either antagonism of a detrimental process/pathway orstimulation of a beneficial process/pathway.

Many immune-related diseases are known and have been extensivelystudied. Such diseases include cancer (neoplasia), immune-mediatedinflammatory diseases, non-immune-mediated inflammatory diseases,infectious diseases, and immunodeficiency diseases.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens that are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells (APCs), virus infected cells,cancer cells, grafts, etc. The T cell system eliminates these alteredcells, which pose a health threat to the host mammal. T cells includehelper T cells and cytotoxic T cells. Helper T cells proliferateextensively following recognition of an antigen-MHC complex on an APC.Helper T cells also secrete a variety of cytokines (i.e., lymphokines),which play a central role in the activation of B cells, cytotoxic Tcells, and a variety of other cells that participate in the immuneresponse.

Regulatory T cells (Treg) are a subset of helper T cells that play acritical role in inhibition of self-reactive immune responses and areoften found in sites of chronic inflammation such as in tumor tissue.Tregs are defined phenotypically by high cell surface expression ofCD25, CLTA4, GITR, and neuropilin-1 (NRP-1), and are under the controlof the transcription factor FOXP3. Tregs perform their suppressivefunction on activated T cells through contact-dependent mechanisms andcytokine production. Tregs also modulate immune responses by directinteraction with ligands on dendritic cells (DCs), such as CD40Lligation and CTLA4 interaction with B7 molecules on DCs that elicits theinduction of indoleamine 2,3-dioxygenase (IDO). DCs are professionalAPCs capable of inducing immunity or tolerance against self or non-selfantigens. DC-expanded Tregs suppress alloreactivity responses in vitro,and when adoptively transferred, appropriate Tregs inhibited diabetes inNODscid mice or experimentally induced asthma. Specific interactions ofligands on DC with Tregs can also abrogate their suppressive function,such as engagement of GITR in mice, suggesting DC may have a pluralisticrole in modulating Treg function.

The molecules CTLA4 and GITR are representative of ligands definedwithin the CD28-B7 and TNF-superfamilies of co-stimulatory/-inhibitorymolecules, respectively. These molecules are highly expressed on Tregsbut are typically upregulated on activated T cells. More recently, aprotein designated TIGIT (for T-cell immunoreceptor with Ig and ITIMdomains) was identified as a cell surface-bound protein specificallyexpressed in T cells that possessing an IgV domain, a transmembranedomain, and two putative immunoreceptor tyrosine inhibitory (ITIM)motifs. TIGIT was shown to be particularly expressed on Treg and memoryT cell subsets, as well as NK cells. As there is an unmet need for newtherapeutics and methods of treatment of immune-related disorders andparticularly cancers, described herein are unexpectedly efficacioustherapeutic compositions, such as the anti-TIGIT antibodies andcompositions thereof, and methods of treatment of immune-relateddisorders and cancers, which involve modulating the interaction of TIGITwith its binding partners.

SUMMARY OF THE INVENTION

The present invention provides anti-TIGIT (T-cell immunoreceptor with Igand ITIM domains) antibodies and variants thereof with improvedproperties, including, for example, binding affinity, cross-reactivity,pharmacokinetics, and/or expression. In particular, the presentinvention provides anti-TIGIT antibodies and variants thereof thatpossess, for example, high binding affinity to human TIGIT;cross-reactivity between human TIGIT, cynomolgus monkey (cyno) TIGIT,and/or rabbit TIGIT; desirable clearance properties in cyno; andbiochemical and biophysical properties that confer the antibodies andvariants thereof with high stability.

In one aspect, the invention features an antibody that specificallybinds to human TIGIT, wherein the antibody binds to an epitope on humanTIGIT comprising one or more of amino acid residues Ser78, Ser80, andLys82 of human TIGIT. In one embodiment, the epitope comprises aminoacid residues Ser80 and Lys82 of human TIGIT. In another embodiment, theepitope comprises amino acid residues Ser78, Ser80, and Lys82 of humanTIGIT. In another embodiment, the epitope further comprises amino acidresidue Ala67 of human TIGIT. In another embodiment, the epitope furthercomprises one or more additional amino acid residues selected from thegroup consisting of Glu60, Leu65, and Ile68 of human TIGIT. In anotherembodiment, the epitope further comprises one or more additional aminoacid residues selected from the group consisting of Gln56, Asn70, Leu73,and His111 of human TIGIT. In another embodiment, the epitope furthercomprises one or more additional amino acid residues selected from thegroup consisting of Thr55, Asn58, Asp63, Gln64, His76, Ile77, and Pro79of human TIGIT. In another embodiment, the epitope consists of aminoacid residues Thr55, Gln56, Asn58, Glu60, Asp63, Gln64, Leu65, Ala67,Ile68, Asn70, Leu73, His76, Ile77, Ser78, Pro79, Ser80, Lys82, andHis111 of human TIGIT.

In another aspect, the invention features an antibody that specificallybinds to human TIGIT, wherein the antibody binds to an epitope on humanTIGIT comprising one or more of amino acid residues Thr55, Ser80, andLys82 of human TIGIT. In one embodiment, the epitope comprises aminoacid residue Lys82 of human TIGIT. In another embodiment, the epitopecomprises amino acid residues Thr55, Ser80, and Lys82 of human TIGIT. Inanother embodiment, the epitope further comprises amino acid residueGln56 of human TIGIT. In another embodiment, the epitope furthercomprises amino acid residue Ile77 or Pro79 of human TIGIT. In anotherembodiment, the epitope further comprises amino acid residues Ile77 andPro79 of human TIGIT. In another embodiment, the epitope furthercomprises amino acid residue Asn58 or Glu60 of human TIGIT. In anotherembodiment, the epitope further comprises amino acid residues Asn58 andGlu60 of human TIGIT. In another embodiment, the epitope furthercomprises one or more additional amino acid residues selected from thegroup consisting of Leu65, Ile68, Leu73, His76, Ser78, and His111 ofhuman TIGIT. In another embodiment, the epitope further comprises aminoacid residues Leu65, Ile68, Leu73, His76, Ser78, and His111 of humanTIGIT. In yet another embodiment, the epitope consists of Thr55, Gln56,Asn58, Glu60, Leu65, Ile68, Leu73, His76, Ile77, Ser78, Pro79, Ser80,Lys82, and His111 of human TIGIT.

In another aspect, the invention features an antibody that specificallybinds to human TIGIT, wherein the antibody comprises a paratopecomprising one or more amino acid residues selected from the groupconsisting of heavy chain variable region amino acid residues Asn32,Tyr52, Arg52b, Phe53, Lys54, Tyr56, Asp58, Tyr99, Asp100, Leu100a,Leu100b, and Ala100c and light chain variable region amino acid residuesTyr27d, Tyr92, Ser93, Thr94, and Phe96. In one embodiment, the paratopeconsists of heavy chain variable region amino acid residues Asn32,Tyr52, Arg52b, Phe53, Lys54, Tyr56, Asp58, Tyr99, Asp100, Leu100a,Leu100b, and Alai 00c and light chain variable region amino acidresidues Tyr27d, Tyr92, Ser93, Thr94, and Phe96. In any of the aboveaspects, the antibody may be capable of binding to rabbit TIGIT.

In another aspect, the invention features an antibody that specificallybinds to human TIGIT, wherein the antibody binds to an epitope on humanTIGIT comprising one or more amino acid residues selected from the groupconsisting of Gln53, His111, and Tyr113 of human TIGIT. In someembodiments, the epitope further comprises Gln56 of human TIGIT. In someembodiments, the epitope further comprises Glu60, Leu65, Ile68, Asn70,Leu73, and His76 of human TIGIT.

In another aspect, the invention features an antibody that specificallybinds to human TIGIT, wherein the antibody comprises the following sixhypervariable regions (HVRs): an HVR-H1 comprising the amino acidsequence of SNSAAWN (SEQ ID NO: 1); an HVR-H2 comprising the amino acidsequence of KTYYRFKWYSDYAVSVKG (SEQ ID NO: 2); an HVR-H3 comprising theamino acid sequence of ESTTYDLLAGPFDY (SEQ ID NO: 3); an HVR-L1comprising the amino acid sequence of KSSQTVLYSSNNKKYLA (SEQ ID NO: 4);an HVR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 5);and an HVR-L3 comprising the amino acid sequence of QQYYSTPFT (SEQ IDNO: 6). In some embodiments, the antibody further comprises thefollowing light chain variable region framework regions (FRs): an FR-L1comprising the amino acid sequence of DIVMTQSPDSLAVSLGERATINC (SEQ IDNO: 7); an FR-L2 comprising the amino acid sequence of WYQQKPGQPPNLLIY(SEQ ID NO: 8); an FR-L3 comprising the amino acid sequence ofGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 9); and an FR-L4 comprisingthe amino acid sequence of FGPGTKVEIK (SEQ ID NO: 10). In someembodiments, the antibody further comprises the following heavy chainvariable region FRs: an FR-H1 comprising the amino acid sequence ofX₁VQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 11), wherein X₁ is Q or E;an FR-H2 comprising the amino acid sequence of WIRQSPSRGLEWLG (SEQ IDNO: 12); an FR-H3 comprising the amino acid sequence ofRITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 13); and an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 14). Insome embodiments, the antibody further comprises the following heavychain variable region FRs: an FR-H1 comprising the amino acid sequenceof EVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 15); an FR-H2 comprisingthe amino acid sequence of WIRQSPSRGLEWLG (SEQ ID NO: 12); an FR-H3comprising the amino acid sequence of RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR(SEQ ID NO: 13); and an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 14). In some embodiments, the antibody furthercomprises the following heavy chain variable region FRs: an FR-H1comprising the amino acid sequence of QVQLQQSGPGLVKPSQTLSLTCAISGDSVS(SEQ ID NO: 16); an FR-H2 comprising the amino acid sequence ofWIRQSPSRGLEWLG (SEQ ID NO: 12); an FR-H3 comprising the amino acidsequence of RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 13); and anFR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 14).In some embodiments, the antibody is capable of binding to rabbit TIGIT.

In another aspect, the invention features an antibody that specificallybinds to human TIGIT, wherein the antibody comprises the following sixHVRs: an HVR-H1 comprising the amino acid sequence of SYPMN (SEQ ID NO:17); an HVR-H2 comprising the amino acid sequence of WINTNTGNPTYVQGFTG(SEQ ID NO: 18); an HVR-H3 comprising the amino acid sequence ofTGGHTYDSYAFDV (SEQ ID NO: 19); an HVR-L1 comprising the amino acidsequence of RASQVISSSLA (SEQ ID NO: 20); an HVR-L2 comprising the aminoacid sequence of AASTLQS (SEQ ID NO: 21); and an HVR-L3 comprising theamino acid sequence of QHLHGYPX₁N (SEQ ID NO: 22), wherein X₁ is C or S.In some embodiments, the antibody comprises the following six HVRs: anHVR-H1 comprising the amino acid sequence of SYPMN (SEQ ID NO: 17); anHVR-H2 comprising the amino acid sequence of WINTNTGNPTYVQGFTG (SEQ IDNO: 18); an HVR-H3 comprising the amino acid sequence of TGGHTYDSYAFDV(SEQ ID NO: 19); an HVR-L1 comprising the amino acid sequence ofRASQVISSSLA (SEQ ID NO: 20); an HVR-L2 comprising the amino acidsequence of AASTLQS (SEQ ID NO: 21); and an HVR-L3 comprising the aminoacid sequence of QHLHGYPSN (SEQ ID NO: 23). In some embodiments, theantibody further comprises the following heavy chain variable regionFRs: an FR-H1 comprising the amino acid sequence ofEVQLVQSGSDLKKPGASVRVSCKASGYTFT (SEQ ID NO: 24); an FR-H2 comprising theamino acid sequence of WVRQAPGHGLEWMG (SEQ ID NO: 25); an FR-H3comprising the amino acid sequence of RFVFSLDTSVNTAYLQISSLKAEDTAVYFCAR(SEQ ID NO: 26); and an FR-H4 comprising the amino acid sequence ofWGQGTMVTVSS (SEQ ID NO: 27). In some embodiments, antibody comprises thefollowing six HVRs: an HVR-H1 comprising the amino acid sequence ofSYPMN (SEQ ID NO: 17); an HVR-H2 comprising the amino acid sequence ofWINTNTGNPTYVQGFTG (SEQ ID NO: 18); an HVR-H3 comprising the amino acidsequence of TGGHTYDSYAFDV (SEQ ID NO: 19); an HVR-L1 comprising theamino acid sequence of RASQVISSSLA (SEQ ID NO: 20); an HVR-L2 comprisingthe amino acid sequence of AASTLQS (SEQ ID NO: 21); and an HVR-L3comprising the amino acid sequence of QHLHGYPCN (SEQ ID NO: 28). In someembodiments, the antibody further comprises the following heavy chainvariable region FRs: an FR-H1 comprising the amino acid sequence ofQVQLVQSGSDLKKPGASVRVSCKASGYTFT (SEQ ID NO: 29); an FR-H2 comprising theamino acid sequence of WVRQAPGHGLEWMG (SEQ ID NO: 25); an FR-H3comprising the amino acid sequence of RFVFSLDTSVNTAYLQISSLKAEDTAVYFCAR(SEQ ID NO: 26); and an FR-H4 comprising the amino acid sequence ofWGQGTMVTVSS (SEQ ID NO: 27). In some embodiments, the antibody furthercomprises the following light chain variable region FRs: an FR-L1comprising the amino acid sequence of DIQLTQSPTFLSASVGDRVTITC (SEQ IDNO: 30); an FR-L2 comprising the amino acid sequence of WYQQNPGKAPKLLIY(SEQ ID NO: 31); an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTEFTLTISSLQPEDFVTYYC (SEQ ID NO: 32); and an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 33).

In another aspect, the invention features an antibody that specificallybinds to human TIGIT, wherein the antibody comprises (a) a heavy chainvariable region (VH) having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 34 or 35; (b) a light chain variable region(VL) having at least 95% sequence identity to the amino acid sequence ofSEQ ID NO: 36; or (c) a heavy chain variable region as in (a) and alight chain variable region as in (b). In some embodiments, antibodycomprises (a) a heavy chain variable region (VH) having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 34; (b) alight chain variable region (VL) having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 36; or (c) a heavy chainvariable region as in (a) and a light chain variable region as in (b).In some embodiments, the antibody comprises (a) a heavy chain variableregion (VH) having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 35; (b) a light chain variable region (VL) havingat least 95% sequence identity to the amino acid sequence of SEQ ID NO:36; or (c) a heavy chain variable region as in (a) and a light chainvariable region as in (b). In some embodiments, the antibody is capableof binding to rabbit TIGIT.

In another aspect, the invention features an antibody that specificallybinds to human TIGIT, wherein the antibody comprises (a) a heavy chainvariable region (VH) having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 37; (b) a light chain variable region (VL)having at least 95% sequence identity to the amino acid sequence of SEQID NO: 38; or (c) a heavy chain variable region as in (a) and a lightchain variable region as in (b).

In yet another aspect, the invention features an antibody thatspecifically binds to human TIGIT, wherein the antibody comprises (a) aheavy chain variable region (VH) having at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 39; (b) a light chain variableregion (VL) having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 40; or (c) a heavy chain variable region as in(a) and a light chain variable region as in (b).

In any one of the aspects described above, the antibody may be capableof binding to both human TIGIT and cynomolgus monkey (cyno) TIGIT, butnot murine TIGIT. In some embodiments, the antibody binds human TIGITwith a Kd of about 10 nM or lower and cyno TIGIT with a Kd of about 10nM or lower. In some embodiments, the antibody binds human TIGIT with aKd of about 0.1 nM to about 1 nM and cyno TIGIT with a Kd of about 0.5nM to about 1 nM. In some embodiments, the antibody binds human TIGITwith a Kd of about 0.1 nM or lower and cyno TIGIT with a Kd of about 0.5nM or lower.

In any one of the aspects described above, the antibody may be anantagonist antibody or an agonist antibody.

In some embodiments, the antagonist antibody specifically binds TIGITand inhibits or blocks TIGIT interaction with poliovirus receptor (PVR).In some embodiments, the antagonist antibody inhibits intracellularsignaling mediated by TIGIT binding to PVR. In some embodiments, theantagonist antibody inhibits or blocks binding of human TIGIT to humanPVR with an IC50 value of 10 nM or lower. In some embodiments, theantagonist antibody inhibits or blocks binding of human TIGIT to humanPVR with an IC50 value of 1 nM to about 10 nM. In some embodiments, theantagonist antibody inhibits or blocks binding of cyno TIGIT to cyno PVRwith an IC50 value of 50 nM or lower. In some embodiments, theantagonist antibody inhibits or blocks binding of cyno TIGIT to cyno PVRwith an IC50 value of 1 nM to about 50 nM. In some embodiments, theantagonist antibody inhibits or blocks binding of cyno TIGIT to cyno PVRwith an IC50 value of 1 nM to about 5 nM.

In some embodiments, the agonist antibody specifically binds TIGIT andstimulates the interaction of PVR with CD226 or CD96. In someembodiments, the agonist antibody specifically binds TIGIT andstimulates the interaction of PVR with CD226 and CD96. In someembodiments, the agonist antibody specifically binds TIGIT andstimulates the interaction of human PVR with human CD226 and human CD96.

In some embodiments, the agonist antibody specifically binds TIGIT andstimulates the interaction of cyno PVR with cyno CD226 and cyno CD96.

In another aspect, the invention features an isolated antibody thatcompetes for binding to TIGIT with an antibody of any one of the aboveaspects.

In another aspect, the invention features an isolated antibody thatbinds to the same epitope as an antibody of any one of the aboveaspects.

In some embodiments of any one of the aspects described above, theantibody is monoclonal. In some embodiments, the antibody is human,humanized, or chimeric. In some embodiments, the antibody is afull-length antibody. In some embodiments, the antibody has a clearancefollowing administration (e.g., injection, e.g., intravenous injection)of less than about 10 ml/kg/day (e.g., about 3 ml/kg/day to about 10ml/kg/day). In some embodiments, the antibody antibody has a clearanceof about 3 ml/kg/day to about 8 ml/kg/day. In some embodiments,administration of the antibody is to a mammal (e.g., a monkey, such as acynomolgus monkey, or a human). In some embodiments, the antibody is anantibody fragment that binds TIGIT. In some embodiments, the antibodyfragment is selected from the group consisting of Fab, Fab′, Fab′-SH,Fv, single chain variable fragment (scFv), and (Fab′)₂ fragments. Insome embodiments, the antibody is an IgG class antibody. In someembodiments, the IgG class antibody is an IgG1 subclass antibody. Insome embodiments, an antibody described herein can be for use as amedicament. In some embodiments, an antibody described herein may be foruse in treating or delaying progression of a cancer in a subject in needthereof. In some embodiments, the cancer is selected from the groupconsisting of a non-small cell lung cancer, a small cell lung cancer, arenal cell cancer, a colorectal cancer, an ovarian cancer, a breastcancer, a pancreatic cancer, a gastric carcinoma, a bladder cancer, anesophageal cancer, a mesothelioma, a melanoma, a head and neck cancer, athyroid cancer, a sarcoma, a prostate cancer, a glioblastoma, a cervicalcancer, a thymic carcinoma, a leukemia, a lymphoma, a myeloma, mycosesfungoides, a merkel cell cancer, and a hematologic malignancy. In someembodiments, an antibody described herein may be for use in treating ordelaying progression of multiple myeloma (MM). In some embodiments, anantibody described herein may be for use in treating or delayingprogression of an immune-related disease in a subject in need thereof.In some embodiments, the immune-related disease is associated with a Tcell dysfunctional disorder. In some embodiments, the T celldysfunctional disorder is characterized by T cell exhaustion. In someembodiments, the immune-related disease is selected from the groupconsisting of unresolved acute infection, chronic infection, and tumorimmunity. In some embodiments, an antibody described herein may be foruse in increasing, enhancing, or stimulating an immune response orfunction in a subject in need thereof.

In another aspect, the invention features a polynucleotide (e.g., anisolated polynucleotide) encoding any of the antibodies describedherein. In another aspect, the invention features a vector (e.g., anexpression vector) comprising the polynucleotide for expressing theantibody. In another aspect, the invention features host cellscomprising the preceding polynucleotides and/or vectors. In someembodiments, the host cell is a eukaryotic (e.g., a mammalian cell). Insome embodiments, the eukaryotic cell is a 293 cell, a Chinese hamsterovary (CHO) cell, a yeast cell, or a plant cell. In some embodiments,the host cell is a prokaryotic cell. In some embodiments, theprokaryotic cell is E. coli.

In another aspect, the invention features a method of producing any ofthe antibodies described herein, the method comprising culturing a hostcell that comprises any of the preceding vectors (e.g., expressionvectors) in a culture medium. In some embodiments, the method furthercomprises recovering the antibody from the host cell or the culturemedium. In some embodiments, the host cell is a eukaryotic cell, forexample, a mammalian cell, for example, a 293 cell, a Chinese hamsterovary (CHO) cell, a yeast cell, or a plant cell. In some embodiments,the host cell is a prokaryotic cell. In some embodiments, theprokaryotic cell is E. coli.

In another aspect, the invention features an immunoconjugate comprisingany one of the antibodies described herein and an agent (e.g., atherapeutic agent, e.g., a cytotoxic agent).

In another aspect, the invention features a composition comprising anantibody described herein. In some embodiments, the composition furthercomprises a pharmaceutically acceptable carrier, excipient, or diluent.In some embodiments, the composition is for diagnostic use (e.g., todetect TIGIT expression levels, e.g., TIGIT protein expression levels).In some embodiments, the composition is a pharmaceutical composition. Insome embodiments, the composition further comprises a PD-1 axis bindingantagonist or an additional therapeutic agent.

In another aspect, the invention features the use of an antibodydescribed herein in the manufacture of a medicament for treating ordelaying progression of a cancer in a subject in need thereof. In someembodiments, the cancer is selected from the group consisting of anon-small cell lung cancer, a small cell lung cancer, a renal cellcancer, a colorectal cancer, an ovarian cancer, a breast cancer, apancreatic cancer, a gastric carcinoma, a bladder cancer, an esophagealcancer, a mesothelioma, a melanoma, a head and neck cancer, a thyroidcancer, a sarcoma, a prostate cancer, a glioblastoma, a cervical cancer,a thymic carcinoma, a leukemia, a lymphoma, a myeloma, mycosesfungoides, a merkel cell cancer, and a hematologic malignancy. In someembodiments, the myeloma is MM. In some embodiments, the medicament isformulated for administration subcutaneously, intravenously,intramuscularly, topically, orally, transdermally, intraperitoneally,intraorbitally, by implantation, by inhalation, intrathecally,intraventricularly, or intranasally. In some embodiments, the subject isa human.

In another aspect, the invention features the use of an antibodydescribed herein in the manufacture of a medicament for treating ordelaying progression of an immune-related disease in a subject in needthereof. In some embodiments, the immune-related disease is associatedwith a T cell dysfunctional disorder. In some embodiments, the T celldysfunctional disorder is characterized by T cell exhaustion. In someembodiments, the immune-related disease is selected from the groupconsisting of unresolved acute infection, chronic infection, and tumorimmunity. In some embodiments, the medicament is formulated foradministration subcutaneously, intravenously, intramuscularly,topically, orally, transdermally, intraperitoneally, intraorbitally, byimplantation, by inhalation, intrathecally, intraventricularly, orintranasally. In some embodiments, the subject is a human.

In another aspect, the invention features the use of an antibodydescribed herein in the manufacture of a medicament for increasing,enhancing, or stimulating an immune response or function in a subject inneed thereof. In some embodiments, the medicament is formulated foradministration subcutaneously, intravenously, intramuscularly,topically, orally, transdermally, intraperitoneally, intraorbitally, byimplantation, by inhalation, intrathecally, intraventricularly, orintranasally. In some embodiments, the subject is a human.

In another aspect, the invention features a method for treating ordelaying progression of a cancer in a subject, the method comprisingadministering to the subject an effective amount of any one or more(e.g., 1, 2, 3, 4, 5, or 6 or more) of the antibodies described herein,thereby treating or delaying the progression of the cancer in thesubject. In some embodiments, the cancer is selected from the groupconsisting of a non-small cell lung cancer, a small cell lung cancer, arenal cell cancer, a colorectal cancer, an ovarian cancer, a breastcancer, a pancreatic cancer, a gastric carcinoma, a bladder cancer, anesophageal cancer, a mesothelioma, a melanoma, a head and neck cancer, athyroid cancer, a sarcoma, a prostate cancer, a glioblastoma, a cervicalcancer, a thymic carcinoma, a leukemia, a lymphoma, a myeloma, mycosesfungoides, a merkel cell cancer, and a hematologic malignancy. In someembodiments, the myeloma is MM.

In another aspect, the invention features a method for treating ordelaying progression of an immune-related disease in a subject, themethod comprising administering to the subject an effective amount ofone or more (e.g., 1, 2, 3, 4, 5, or 6 or more) of the antibodiesdescribed herein, thereby treating or delaying the progression of theimmune-related disease in the subject. In some embodiments, theimmune-related disease is associated with a T cell dysfunctionaldisorder. In some embodiments, the T cell dysfunctional disorder ischaracterized by T cell exhaustion. In some embodiments, theimmune-related disease is selected from the group consisting ofunresolved acute infection, chronic infection, and tumor immunity.

In another aspect, the invention features a method of increasing,enhancing, or stimulating an immune response or function in a subject,the comprising administering to the subject an effective amount of oneor more (e.g., 1, 2, 3, 4, 5, or 6 or more) of the antibodies describedherein, thereby increasing, enhancing, or stimulating an immune responseor function in the subject. In some embodiments, the method furthercomprises administering to the subject a PD-1 axis binding antagonist.In some embodiments, the PD-1 axis binding antagonist is administeredprior to or subsequent to the administration of the antibody. In someembodiments, the PD-1 axis binding antagonist is administeredconcurrently with the antibody. In some embodiments, the PD-1 axisbinding antagonist is selected from the group consisting of a PD-1binding antagonist, a PD-L1 binding antagonist, and a PD-L2 bindingantagonist. In some embodiments, the PD-1 axis binding antagonist is aPD-1 binding antagonist. In some embodiments, the PD-1 bindingantagonist inhibits the binding of PD-1 to its ligand binding partners.In some embodiments, the PD-1 binding antagonist inhibits the binding ofPD-1 to PD-L1. In some embodiments, the PD-1 binding antagonist inhibitsthe binding of PD-1 to PD-L2. In some embodiments, the PD-1 bindingantagonist is an anti-PD-1 antibody. In some embodiments, the PD-1binding antagonist is selected from the group consisting of MDX 1106(nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680(AMP-514), PDR001, REGN2810, and BGB-108. In some embodiments, the PD-1axis binding antagonist is a PD-L1 binding antagonist. In someembodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits thebinding of PD-L1 to B7-1. In some embodiments, the PD-L1 bindingantagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. In someembodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. Insome embodiments, the anti-PD-L1 antibody is selected from the groupconsisting of: MPDL3280A (atezolizumab), YW243.55.S70, MDX-1105,MED14736 (durvalumab), and MSB0010718C (avelumab). In some embodiments,the antibody is MPDL3280A. In some embodiments, the PD-1 axis bindingantagonist is a PD-L2 binding antagonist. In some embodiments, the PD-L2binding antagonist is an anti-PD-L2 antibody. In some embodiments, thePD-L2 binding antagonist is an immunoadhesin.

In some embodiments, any one of the methods of treatment described abovemay further comprise administering to the subject an OX40 bindingagonist. In some embodiments, the OX40 binding agonist is administeredprior to or subsequent to the administration of the antibody and/or thePD-1 axis binding antagonist. In some embodiments, the OX40 bindingagonist is administered concurrently with the antibody and/or the PD-1axis binding antagonist. In some embodiments, the OX40 binding agonistis selected from the group consisting of an OX40 agonist antibody, anOX40L agonist fragment, an OX40 oligomeric receptor, and an OX40immunoadhesin. In some embodiments, the OX40 agonist antibody depletescells that express human OX40. In some embodiments, the cells thatexpress human OX40 are CD4+ effector T cells. In some embodiments, thecells that express human OX40 are regulatory T (Treg) cells. In someembodiments, the depleting is by ADCC and/or phagocytosis. In someembodiments, the OX40 agonist antibody binds human OX40 with an affinityof less than or equal to about 1 nM. In some embodiments, the OX40agonist antibody binds human OX40 with an affinity of less than or equalto about 0.45 nM. In some embodiments, the OX40 agonist antibody bindshuman OX40 with an affinity of less than or equal to about 0.4 nM. Insome embodiments, the OX40 agonist antibody binds human OX40 with anEC50 of less than or equal to 0.3 μg/ml. In some embodiments, the OX40agonist antibody binds human OX40 with an EC50 of less than or equal to0.2 μg/ml. In some embodiments, the OX40 agonist antibody increases CD4+effector T cell proliferation and/or increases cytokine production bythe CD4+ effector T cell as compared to proliferation and/or cytokineproduction prior to treatment with the OX40 agonist antibody. In someembodiments, the OX40 agonist antibody increases memory T cellproliferation and/or cytokine production by a memory T cell. In someembodiments, the cytokine production is IFN-γ production. In someembodiments, the OX40 agonist antibody inhibits Treg function. In someembodiments, the OX40 agonist antibody inhibits Treg suppression ofeffector T cell function (e.g., effector T cell proliferation and/orcytokine production). In some embodiments, the effector T cell is a CD4+effector T cell. In some embodiments, the OX40 agonist antibodyincreases OX40 signal transduction in a target cell that expresses OX40.In some embodiments, the OX40 signal transduction is detected bymonitoring NFkB downstream signaling. In some embodiments, the the OX40agonist antibody comprises a variant IgG1 Fc polypeptide comprising amutation that eliminates binding to human effector cells and hasdiminished activity relative to the OX40 agonist antibody comprising anative sequence IgG1 Fc portion. In some embodiments, the OX40 agonistantibody comprises a variant IgG1 Fc polypeptide comprising a DANAmutation. In some embodiments, the OX40 agonist antibody comprises (a) aVH domain comprising (i) a HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 278, 279, or 280, (ii) a HVR-H2 comprising the amino acidsequence of SEQ ID NO: 281, 282, 283, 284, 285, or 286, and (iii) aHVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 287,288, or 289; and (b) a VL domain comprising (i) a HVR-L1 comprising theamino acid sequence of SEQ ID NO: 290, (ii) a HVR-L2 comprising theamino acid sequence of SEQ ID NO: 291, and (iii) a HVR-L3 comprising theamino acid sequence of SEQ ID NO: 292, 293, 294, 295, 296, 297, 298, or299. In some embodiments, the OX40 agonist antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 278; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 281; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 287; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 290; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 291; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO: 292. In someembodiments, the OX40 agonist antibody comprises (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 278; (b) HVR-H2 comprising theamino acid sequence of SEQ ID NO: 281; (c) HVR-H3 comprising the aminoacid sequence of SEQ ID NO: 287; (d) HVR-L1 comprising the amino acidsequence of SEQ ID NO: 290; (e) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 291; and (f) HVR-L3 comprising an amino acidsequence selected from SEQ ID NO: 297. In some embodiments, the OX40agonist antibody comprises (a) HVR-H1 comprising the amino acid sequenceof SEQ ID NO: 278; (b) HVR-H2 comprising the amino acid sequence of SEQID NO: 281; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:287; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 290;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 291; and (f)HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 298.In some embodiments, the OX40 agonist antibody comprises a VH sequencehaving at least 90% sequence identity to the amino acid sequence of anyone of SEQ ID NOs: 300-325. In some embodiments, the OX40 agonistantibody comprises a VH sequence having at least 90% sequence identityto the amino acid sequence of SEQ ID NO: 300. In some embodiments, atotal of 1 to 10 amino acids have been substituted, inserted, and/ordeleted in SEQ ID NO: 300. In some embodiments, the OX40 agonistantibody comprises a VH comprising one, two, or three HVRs selectedfrom: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 278,(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 281, and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 287. In someembodiments, the OX40 agonist antibody comprises a VL sequence having atleast 90% sequence identity to the amino acid sequence of any one of SEQID NOs: 326-351. In some embodiments, the OX40 agonist antibodycomprises a VL having at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 326. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 326. In some embodiments, the OX40 agonist antibody comprises a VLcomprising one, two, or three HVRs selected from (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 290; (b) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 291; and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 292. In some embodiments, the OX40agonist antibody comprises (a) a VH sequence of SEQ ID NO: 300; (b) a VLsequence of SEQ ID NO: 326; or (c) a VH sequence as in (a) and a VLsequence as in (b). In some embodiments, the OX40 agonist antibodycomprises (a) a VH sequence of SEQ ID NO: 319; (b) a VL sequence of SEQID NO: 345; or (c) a VH sequence as in (a) and a VL sequence as in (b).In some embodiments, the OX40 agonist antibody comprises (a) a VHsequence of SEQ ID NO: 320; (b) a VL sequence of SEQ ID NO: 346; or (c)a VH sequence as in (a) and a VL sequence as in (b). In someembodiments, the OX40 agonist antibody is antibody L106, antibody ACT35,MED16469, or MED10562. In some embodiments, the OX40 agonist antibody isa full-length IgG1 antibody. In some embodiments, the OX40 immunoadhesinis a trimeric OX40-Fc protein.

In some embodiments, any one of the methods of treatment described abovemay further comprise administering to the subject an agent thatdecreases or inhibits one or more additional immune co-inhibitoryreceptors. In some embodiments, the one or more additional immuneco-inhibitory receptor is selected from the group consisting of PD-1,CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, and CD96. In some embodiments,any one of the methods of treatment described above may further compriseadministering to the subject an additional therapeutic agent. In someembodiments, the additional therapeutic agent is a chemotherapeuticagent. In some embodiments, antibody is administered subcutaneously,intravenously, intramuscularly, topically, orally, transdermally,intraperitoneally, intraorbitally, by implantation, by inhalation,intrathecally, intraventricularly, or intranasally. In some embodiments,the subject is a human.

In another aspect, the invention features a kit comprising any one ormore (e.g., 1, 2, 3, 4, 5, or 6 or more) of the antibodies describedherein and package insert comprising instructions for using the antibodyfor treating or delaying progression of a cancer in a subject. In someembodiments, the cancer is selected from the group consisting of anon-small cell lung cancer, a small cell lung cancer, a renal cellcancer, a colorectal cancer, an ovarian cancer, a breast cancer, apancreatic cancer, a gastric carcinoma, a bladder cancer, an esophagealcancer, a mesothelioma, a melanoma, a head and neck cancer, a thyroidcancer, a sarcoma, a prostate cancer, a glioblastoma, a cervical cancer,a thymic carcinoma, a leukemia, a lymphoma, a myeloma, mycosesfungoides, a merkel cell cancer, and a hematologic malignancy. In someembodiments, the subject is a human. In some embodiments, the myeloma isMM.

In another aspect, the invention features a kit comprising any one ormore (e.g., 1, 2, 3, 4, 5, or 6 or more) of the antibodies describedherein and a package insert comprising instructions for using theantibody for treating or delaying progression of an immune-relateddisease in a subject. In some embodiments, the immune-related disease isassociated with a T cell dysfunctional disorder. In some embodiments,the T cell dysfunctional disorder is characterized by T cell exhaustion.In some embodiments, the immune-related disease is selected from thegroup consisting of unresolved acute infection, chronic infection, andtumor immunity. In some embodiments, the subject is a human.

In another aspect, the invention features a kit comprising any one ormore (e.g., 1, 2, 3, 4, 5, or 6 or more) of the antibodies describedherein and a package insert comprising instructions for increasing,enhancing, or stimulating an immune response or function in a subject.In some embodiments, the subject is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains at least one drawing executed in color.Copies of this patent or patent application with color drawings will beprovided by the Office upon request and payment of the necessary fee.

FIG. 1A is a schematic diagram of a CHO-TIGIT binding assay.

FIG. 1B is a graph showing the results of the CHO-TIGIT binding assaydepicted in FIG. 1A using human TIGIT-expressing CHO cells and theindicated OMT-derived anti-TIGIT antibodies at varying concentrations,as analyzed by FACS.

FIG. 1C is a graph showing the results of the CHO-TIGIT binding assaydepicted in FIG. 1A using cynomolgus monkey (cyno) TIGIT-expressing CHOcells and the indicated OMT-derived anti-TIGIT antibodies at varyingconcentrations, as analyzed by FACS.

FIG. 1D is a graph showing the results of the CHO-TIGIT binding assaydepicted in FIG. 1A using human TIGIT-expressing CHO cells and theindicated SD-derived anti-TIGIT antibodies at varying concentrations, asanalyzed by FACS.

FIG. 1E is a graph showing the results of the CHO-TIGIT binding assaydepicted in FIG. 1A using cyno TIGIT-expressing CHO cells and theindicated SD-derived anti-TIGIT antibodies at varying concentrations, asanalyzed by FACS.

FIG. 2A is a graph showing the binding of the indicated OMT-derivedanti-TIGIT antibodies to human CD4 T cells as a function of antibodyconcentration.

FIG. 2B is a graph showing the binding of the indicated OMT-derivedanti-TIGIT antibodies to human CD8 T cells as a function of antibodyconcentration.

FIG. 2C is a graph showing the binding of the indicated SD-derivedanti-TIGIT antibodies to human CD4 T cells as a function of antibodyconcentration.

FIG. 2D is a graph showing the binding of the indicated SD-derivedanti-TIGIT antibodies to human CD8 T cells as a function of antibodyconcentration.

FIG. 3A is a graph showing the results of a blocking ELISA assay usinghuman PVR-Fc fusion protein coated-plates, human TIGIT, and theindicated anti-TIGIT antibodies (10A7 and 4.1D3.Q1E in Fab or IgGformat) or anti-CSF1 Fab control, as a function of antibody/Fabconcentration.

FIG. 3B is a graph showing the results of a blocking ELISA assay usingcyno PVR-Fc fusion protein coated-plates, cyno TIGIT, and the indicatedanti-TIGIT antibodies (10A7 and 4.1D3.Q1E in Fab or IgG format) oranti-CSF1 Fab control, as a function of antibody/Fab concentration.

FIG. 3C is a graph showing the results of a blocking ELISA assay usingmurine PVR-Fc fusion protein coated-plates, murine TIGIT, and theindicated anti-TIGIT antibodies (10A7 and 4.1D3.Q1E in Fab or IgGformat) or anti-CSF1 Fab control, as a function of antibody/Fabconcentration.

FIG. 3D is a table showing the results of the blocking ELISA assaysdescribed in FIGS. 3A-3C.

FIG. 4A is a schematic diagram of a CHO-TIGIT PVR blocking assay.

FIG. 4B is a graph showing the results of the CHO-TIGIT PVR blockingassay depicted in FIG. 4A using human TIGIT-expressing CHO cells and theindicated OMT-derived anti-TIGIT antibodies at varying concentrations,as analyzed by FACS.

FIG. 4C is a graph showing the results of the CHO-TIGIT PVR blockingassay depicted in FIG. 4A using cyno TIGIT-expressing CHO cells and theindicated OMT-derived anti-TIGIT antibodies at varying concentrations,as analyzed by FACS.

FIG. 4D is a graph showing the results of the CHO-TIGIT PVR blockingassay depicted in FIG. 4A using human TIGIT-expressing CHO cells and theindicated SD-derived anti-TIGIT antibodies at varying concentrations, asanalyzed by FACS.

FIG. 4E is a graph showing the results of the CHO-TIGIT PVR blockingassay depicted in FIG. 4A using cyno TIGIT-expressing CHO cells and theindicated SD-derived anti-TIGIT antibodies at varying concentrations, asanalyzed by FACS.

FIG. 5A is a graph showing the pharmacokinetic clearance of theindicated anti-TIGIT antibodies in cyno following 10 mg/kg intravenousadministration as a function of serum concentration (μg/ml) over time(days).

FIG. 5B is a table showing the calculated clearance values for eachantibody tested in the pharmacokinetic clearance experiments in FIG. 5A.

FIG. 5C is a graph showing the pharmacokinetic clearance of anti-TIGITantibodies h10A7.K4G3 and 4.1D3 in cyno following 10 mg/kg intravenousadministration as a function of serum concentration (μg/ml) over time(days; d0-d7).

FIG. 5D is a table showing the calculated clearance values for theanti-TIGIT antibodies h10A7.K4G3 and 4.1D3 tested in the pharmacokineticclearance experiments in FIG. 5C.

FIG. 6A is a rendering of the crystal structure of the 4.1D3 Fab boundto human TIGIT, with the 4.1D3 heavy chain (HC) and light chain (LC)regions indicated and distinguished by color.

FIG. 6B is a rendering of the crystal structure of the 4.1D3 Fab boundto human TIGIT, superimposed with the known PVR-TIGIT structure (PDB3UDW), showing that the 4.1D3 antibody and PVR have overlapping bindingsites for TIGIT.

FIG. 6C is a rendering of the crystal structure of the 4.1D3 Fab boundto human TIGIT, with the relative location of the PVR binding siteindicated. Also indicated is the relative location of the 72-79 loop ofTIGIT.

FIGS. 6D and 6E are renderings of the crystal structure of the 4.1D3 Fabbound to human TIGIT from different views, identifying certain keycontact residues of 4.1D3 (paratope residues) and TIGIT (epitoperesidues). The boxed residues indicated in FIG. 6E were identified asfunctionally important epitopic residues by alanine scanningmutagenesis.

FIG. 7A is a graph of showing the K_(D) values (nM) for each of theidentified anti-TIGIT antibodies for the indicated human TIGIT alaninescanning mutant.

FIG. 7B is a graph of showing the K_(D) values (nM) for each of theidentified anti-TIGIT antibodies for the indicated human TIGIT alaninescanning mutant, normalized against the wild-type human TIGIT target.Alanine scanning mutants of TIGIT that resulted in a 1- to 10-fold orgreater than 10-fold drop in binding affinity are indicated by lightgray and dark gray shading, respectively.

FIG. 7C is a ribbon rendering of the human TIGIT structure with residuesidentified as important for 10A7 recognition by alanine scanningmutagenesis indicated and represented as spheres.

FIG. 7D is a ribbon rendering of the human TIGIT structure with residuesidentified as important for h1A5 recognition by alanine scanningmutagenesis indicated and represented as spheres.

FIG. 7E is a ribbon rendering of the human TIGIT structure with residuesidentified as important for 4.1D3 recognition by alanine scanningmutagenesis indicated and represented as spheres.

FIG. 7F is a ribbon rendering of the human TIGIT structure with residuesidentified as important for 7.4A3 recognition by alanine scanningmutagenesis indicated and represented as spheres.

FIG. 7G is a ribbon rendering of the human TIGIT structure with residuesidentified as important for 4.1A4 recognition by alanine scanningmutagenesis indicated and represented as spheres.

FIG. 7H is a ribbon rendering of the human TIGIT structure with residuesidentified as important for h6B2 recognition by alanine scanningmutagenesis indicated and represented as spheres.

FIG. 7I is a ribbon rendering of the human TIGIT structure with residuesidentified as important for h7E7 recognition by alanine scanningmutagenesis indicated and represented as spheres.

FIG. 8 is a rendering of the crystal structure of the 1A5 Fab bound tohuman TIGIT, with the 1A5 heavy chain (HC) and light chain (LC) regionsindicated and distinguished by color.

FIG. 9 is a rendering of the crystal structures of the 4.1D3-TIGIT,1A5-TIGIT, and 10A7-TIGIT complexes, superimposed on one another withrespect to TIGIT, showing that the three anti-TIGIT antibodies bind tohuman TIGIT at non-identical epitopes.

FIG. 10A is a rendering of the crystal structure of the 10A7 Fab boundto human TIGIT, with the 10A7 heavy chain (HC) and light chain (LC)regions indicated and distinguished by color.

FIG. 10B is a rendering of the crystal structure of the 10A7 Fab boundto human TIGIT, with the relative location of the PVR binding siteindicated. Also indicated is the relative location of the 72-79 loop ofTIGIT.

FIG. 11 is a series of renderings of the 4.1D3 (top left), 10A7 (centermiddle), and 1A5 (bottom right) antibodies and the relative location ofthe Ser78, Ser80, and Lys82 residues of human TIGIT. The structuresindicate that the Ser78, Ser80, and Lys82 residues of human TIGIT arekey epitopic residues for 4.1D3, but not for 10A7 or 1A5, where thesethree residues are located at a distance further from the antibodybinding pocket.

FIG. 12 is a series of graphs showing CD45, PD-1, TIGIT, and CD226expression on CD4+ and CD8+ T cells isolated from the bone marrow ofmultiple myeloma (MM) patients (top row) and PD-1, TIGIT, and CD226expression on CD4+ and CD8+ T cells isolated from the peripheral bloodof healthy patients (bottom row), as assessed by multi-color flowcytometry.

FIG. 13 is a series of flow cytometry graphs (left) and accompanyingplots (right) showing that TIGIT and PD-1 are co-expressed on CD4+ andCD8+ T cells in bone marrow of MM patients.

DETAILED DESCRIPTION OF THE INVENTION I. General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3d edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., (2003)); the seriesMethods in Enzymology (Academic Press, Inc.): PCR 2: A PracticalApproach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and AnimalCell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J. B. LippincottCompany, 1993).

II. Definitions

The term “TIGIT” or “T-cell immunoreceptor with Ig and ITIM domains” asused herein refers to any native TIGIT from any vertebrate source,including mammals such as primates (e.g., humans) and rodents (e.g.,mice and rats), unless otherwise indicated. TIGIT is also known in theart as DKFZp667A205, FLJ39873, V-set and immunoglobulindomain-containing protein 9, V-set and transmembrane domain-containingprotein 3, VSIG9, VSTM3, and WUCAM. The term encompasses “full-length,”unprocessed TIGIT (e.g., full-length human TIGIT having the amino acidsequence of SEQ ID NO: 352), as well as any form of TIGIT that resultsfrom processing in the cell (e.g., processed human TIGIT without asignal sequence, having the amino acid sequence of SEQ ID NO: 353). Theterm also encompasses naturally occurring variants of TIGIT, e.g.,splice variants or allelic variants. The amino acid sequence of anexemplary human TIGIT may be found under UniProt Accession NumberQ495A1.

The terms “anti-TIGIT antibody” and “an antibody that specifically bindsto TIGIT” refer to an antibody that is capable of binding TIGIT withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting TIGIT. In one embodiment, theextent of binding of an anti-TIGIT antibody to an unrelated, non-TIGITprotein is less than about 10% of the binding of the antibody to TIGITas measured, e.g., by a radioimmunoassay (RIA). In certain embodiments,an antibody that binds to TIGIT has a dissociation constant (Kd) of ≤1μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M).In certain embodiments, an anti-TIGIT antibody binds to an epitope ofTIGIT that is conserved among TIGIT from different species or an epitopeon TIGIT that allows for cross-species reactivity, such as an epitopecomprising amino acid residues Ser78, Ser80, and Lys82.

The term “antibody” includes monoclonal antibodies (including fulllength antibodies which have an immunoglobulin Fc region), antibodycompositions with polyepitopic specificity, multispecific antibodies(e.g., bispecific antibodies, diabodies, and single-chain molecules, aswell as antibody fragments (e.g., Fab, F(ab′)₂, and Fv). The term“immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

The term an “isolated antibody” when used to describe the variousantibodies disclosed herein, means an antibody that has been identifiedand separated and/or recovered from a cell or cell culture from which itwas expressed. Contaminant components of its natural environment arematerials that would typically interfere with diagnostic or therapeuticuses for the polypeptide, and can include enzymes, hormones, and otherproteinaceous or non-proteinaceous solutes. In some embodiments, anantibody is purified to greater than 95% or 99% purity as determined by,for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing(IEF), capillary electrophoresis) or chromatographic (e.g., ion exchangeor reverse phase HPLC). For a review of methods for assessment ofantibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87(2007). In preferred embodiments, the antibody will be purified (1) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (2)to homogeneity by SDS-PAGE under non-reducing or reducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes antibodies in situ within recombinant cells, because at leastone component of the polypeptide natural environment will not bepresent. Ordinarily, however, isolated polypeptide will be prepared byat least one purification step.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. An IgM antibody consists of 5 of the basic heterotetramer unitsalong with an additional polypeptide called a J chain, and contains 10antigen binding sites, while IgA antibodies comprise from 2-5 of thebasic 4-chain units which can polymerize to form polyvalent assemblagesin combination with the J chain. In the case of IgGs, the 4-chain unitis generally about 150,000 Daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for μ and ε isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain at its other end. The V_(L) is aligned with the V_(H) and theC_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see, e.g., Basic and Clinical Immunology, 8th Edition,Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton &Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa and lambda, based on the amino acid sequences of theirconstant domains. Depending on the amino acid sequence of the constantdomain of their heavy chains (CH), immunoglobulins can be assigned todifferent classes or isotypes. There are five classes ofimmunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chainsdesignated α, δ, ε, γ, and μ, respectively. The γ and α classes arefurther divided into subclasses on the basis of relatively minordifferences in the CH sequence and function, e.g., humans express thefollowing subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domains of the heavy chain and light chain may be referred toas “VH” and “VL”, respectively. These domains are generally the mostvariable parts of the antibody (relative to other antibodies of the sameclass) and contain the antigen binding sites.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines the specificity of aparticular antibody for its particular antigen. However, the variabilityis not evenly distributed across the entire span of the variabledomains. Instead, it is concentrated in three segments calledhypervariable regions (HVRs) both in the light-chain and the heavy chainvariable domains. The more highly conserved portions of variable domainsare called the framework regions (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three HVRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The HVRs in each chain are held together in close proximity by the FRregions and, with the HVRs from the other chain, contribute to theformation of the antigen binding site of antibodies (see Kabat et al.,Sequences of Immunological Interest, Fifth Edition, National Instituteof Health, Bethesda, Md. (1991)). The constant domains are not involveddirectly in the binding of antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

A “blocking antibody” or an “antagonist antibody” is one that inhibitsor reduces a biological activity of the antigen it binds. In someembodiments, blocking antibodies or antagonist antibodies substantiallyor completely inhibit the biological activity of the antigen. Theanti-TIGIT antibodies of the invention may block signaling through PVR,PVRL2, and/or PVRL3 so as to restore a functional response by T-cells(e.g., proliferation, cytokine production, target cell killing) from adysfunctional state to antigen stimulation.

An “agonist antibody” or “activating antibody” is one that enhances orinitiates signaling by the antigen to which it binds. In someembodiments, agonist antibodies cause or activate signaling without thepresence of the natural ligand. The OX40 agonist antibodies of theinvention may increase memory T cell proliferation, increase cytokineproduction by memory T cells, inhibit Treg cell function, and/or inhibitTreg cell suppression of effector T cell function, such as effector Tcell proliferation and/or cytokine production.

An “epitope” is the portion of the antigen to which the antibodyspecifically binds. For a polypeptide antigen, the epitope is generallya peptide portion of about 4-15 amino acid residues, which may becontiguous or non-contiguous.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

By “paratope” is meant the part of an antibody which selectively bindsthe epitope of an antigen. The paratope typically includes amino acidsfrom the antibody's VH and VL chains, and, in particular, from theantibody's HVR region(s).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations and/orpost-translation modifications (e.g., isomerizations, amidations) thatmay be present in minor amounts. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site. In contrast topolyclonal antibody preparations which typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by a variety of techniques,including, for example, the hybridoma method (e.g., Kohler andMilstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (ColdSpring Harbor Laboratory Press, 2^(nd) ed. 1988); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),phage-display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhuet al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol.340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004), and technologies for producing human or human-likeantibodies in animals that have parts or all of the human immunoglobulinloci or genes encoding human immunoglobulin sequences (see, e.g., WO1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits etal., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al.,Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33(1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and U.S. Pat. No. 5,661,016; Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison,Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); andLonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The term “naked antibody” refers to an antibody that is not conjugatedto a cytotoxic moiety or radiolabel.

The terms “full-length antibody,” “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody in its substantiallyintact form, as opposed to an antibody fragment. Specifically wholeantibodies include those with heavy and light chains including an Fcregion. The constant domains may be native sequence constant domains(e.g., human native sequence constant domains) or amino acid sequencevariants thereof. In some cases, the intact antibody may have one ormore effector functions.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen-binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules and multispecific antibodies formed fromantibody fragments. Papain digestion of antibodies produced twoidentical antigen-binding fragments, called “Fab” fragments, and aresidual “Fc” fragment, a designation reflecting the ability tocrystallize readily. The Fab fragment consists of an entire L chainalong with the variable region domain of the H chain (V_(H)), and thefirst constant domain of one heavy chain (C_(H)1). Each Fab fragment ismonovalent with respect to antigen binding, i.e., it has a singleantigen-binding site. Pepsin treatment of an antibody yields a singlelarge F(ab′)₂ fragment which roughly corresponds to two disulfide linkedFab fragments having different antigen-binding activity and is stillcapable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having a few additional residues at the carboxy terminus ofthe C_(H)1 domain including one or more cysteines from the antibodyhinge region. Fab′-SH is the designation herein for Fab′ in which thecysteine residue(s) of the constant domains bear a free thiol group.F(ab′)₂ antibody fragments originally were produced as pairs of Fab′fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, the region which is alsorecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of the sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

“Functional fragments” of the antibodies of the invention comprise aportion of an intact antibody, generally including the antigen bindingor variable region of the intact antibody or the Fc region of anantibody which retains or has modified FcR binding capability. Examplesof antibody fragments include linear antibody, single-chain antibodymolecules and multispecific antibodies formed from antibody fragments.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10) residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,thereby resulting in a bivalent fragment, i.e., a fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described in greater detail in, for example, EP 404,097; WO93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993).

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is(are) identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855(1984)). Chimeric antibodies of interest herein include PRIMATIZED®antibodies wherein the antigen-binding region of the antibody is derivedfrom an antibody produced by, e.g., immunizing macaque monkeys with anantigen of interest. As used herein, “humanized antibody” is used asubset of “chimeric antibodies.”

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from an HVR(hereinafter defined) of the recipient are replaced by residues from anHVR of a non-human species (donor antibody) such as mouse, rat, rabbitor non-human primate having the desired specificity, affinity, and/orcapacity. In some instances, framework (“FR”) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications may be made to further refine antibody performance, suchas binding affinity. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin sequence, and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence, although the FR regions may include one or more individual FRresidue substitutions that improve antibody performance, such as bindingaffinity, isomerization, immunogenicity, etc. The number of these aminoacid substitutions in the FR are typically no more than 6 in the Hchain, and in the L chain, no more than 3. The humanized antibodyoptionally will also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op.Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani andHamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris,Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr.Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is an antibody that possesses an amino-acid sequencecorresponding to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk, J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

The expression “variable-domain residue-numbering as in Kabat” or“amino-acid-position numbering as in Kabat,” and variations thereof,refers to the numbering system used for heavy-chain variable domains orlight-chain variable domains of the compilation of antibodies in Kabatet al., supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy-chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy-chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

“Framework” or “FR” residues are those variable-domain residues otherthan the HVR residues as herein defined.

A “human consensus framework” or “acceptor human framework” is aframework that represents the most commonly occurring amino acidresidues in a selection of human immunoglobulin VL or VH frameworksequences. Generally, the selection of human immunoglobulin VL or VHsequences is from a subgroup of variable domain sequences. Generally,the subgroup of sequences is a subgroup as in Kabat et al., Sequences ofProteins of Immunological Interest, 5^(th) Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991). Examples includefor the VL, the subgroup may be subgroup kappa I, kappa II, kappa III orkappa IV as in Kabat et al., supra. Additionally, for the VH, thesubgroup may be subgroup I, subgroup II, or subgroup III as in Kabat etal., supra. Alternatively, a human consensus framework can be derivedfrom the above in which particular residues, such as when a humanframework residue is selected based on its homology to the donorframework by aligning the donor framework sequence with a collection ofvarious human framework sequences. An acceptor human framework “derivedfrom” a human immunoglobulin framework or a human consensus frameworkmay comprise the same amino acid sequence thereof, or it may containpre-existing amino acid sequence changes. In some embodiments, thenumber of pre-existing amino acid changes are 10 or less, 9 or less, 8or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 orless.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al., supra. In one embodiment, the VH subgroup III consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (HC-FR1) (SEQID NO: 229); WVRQAPGKGLEWV (HC-FR2) (SEQ ID NO: 230);RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (HC-FR3) (SEQ ID NO: 232); andWGQGTLVTVSA (HC-FR4) (SEQ ID NO: 232).

A “VL kappa I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al., supra. In one embodiment, the VH subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences: DIQMTQSPSSLSASVGDRVTITC (LC-FR1) (SEQID NO: 233); WYQQKPGKAPKLLIY (LC-FR2) (SEQ ID NO: 234);GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (LC-FR3) (SEQ ID NO: 235); andFGQGTKVEIKR (LC-FR4) (SEQ ID NO: 236).

An “amino-acid modification” at a specified position, for example, ofthe Fc region, refers to the substitution or deletion of the specifiedresidue, or the insertion of at least one amino acid residue adjacentthe specified residue. Insertion “adjacent” to a specified residue meansinsertion within one to two residues thereof. The insertion may beN-terminal or C-terminal to the specified residue. The preferred aminoacid modification herein is a substitution.

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen, e.g., TIGIT).Unless indicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Specificillustrative and exemplary embodiments for measuring binding affinityare described in the following.

An “affinity-matured” antibody is one with one or more alterations inone or more HVRs thereof that result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody that does notpossess those alteration(s). In one embodiment, an affinity-maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity-matured antibodies are produced by procedures known inthe art. For example, Marks et al., Bio/Technology 10:779-783 (1992)describes affinity maturation by VH- and VL-domain shuffling. Randommutagenesis of HVR and/or framework residues is described by, forexample: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994);Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol.155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995);and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

As used herein, the term “binds,” “specifically binds to,” or is“specific for” refers to measurable and reproducible interactions suchas binding between a target and an antibody, which is determinative ofthe presence of the target in the presence of a heterogeneous populationof molecules including biological molecules. For example, an antibodythat specifically binds to a target (which can be an epitope) is anantibody that binds this target with greater affinity, avidity, morereadily, and/or with greater duration than it binds to other targets. Inone embodiment, the extent of binding of an antibody to an unrelatedtarget is less than about 10% of the binding of the antibody to thetarget as measured, for example, by a radioimmunoassay (RIA). In certainembodiments, an antibody that specifically binds to a target has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM.In certain embodiments, an antibody specifically binds to an epitope ona protein that is conserved among the protein from different species. Inanother embodiment, specific binding can include, but does not requireexclusive binding. The term as used herein can be exhibited, forexample, by a molecule having a Kd for the target of 10⁻⁴M or lower,alternatively 10⁻⁵M or lower, alternatively 10⁻⁶ M or lower,alternatively 10⁻⁷ M or lower, alternatively 10⁻⁸ M or lower,alternatively 10⁻⁹ M or lower, alternatively 10⁻¹⁰ M or lower,alternatively 10⁻¹¹ M or lower, alternatively 10⁻¹² M or lower or a Kdin the range of 10⁻⁴ M to 10⁻⁶ M or 10⁻⁶ M to 10⁻¹⁰ M or 10⁻⁷ M to 10⁻⁹M. As will be appreciated by the skilled artisan, affinity and Kd valuesare inversely related. A high affinity for an antigen is measured by alow Kd value. In one embodiment, the term “specific binding” refers tobinding where a molecule binds to a particular polypeptide or epitope ona particular polypeptide without substantially binding to any otherpolypeptide or polypeptide epitope.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2 (including IgG2A and IgG2B), IgG-3, or IgG-4 subtypes, IgA(including IgA-1 and IgA-2), IgE, IgD or IgM. The Ig fusions preferablyinclude the substitution of a domain of a polypeptide or antibodydescribed herein in the place of at least one variable region within anIg molecule. In a particularly preferred embodiment, the immunoglobulinfusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3regions of an IgG1 molecule. For the production of immunoglobulinfusions see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995. Forexample, useful immunoadhesins for combination therapy herein includepolypeptides that comprise the extracellular or OX40 binding portions ofOX40L or the extracellular or OX40L binding portions of OX40, fused to aconstant domain of an immunoglobulin sequence, such as a OX40 ECD-Fc ora OX40L ECD-Fc. Immunoadhesin combinations of Ig Fc and ECD of cellsurface receptors are sometimes termed soluble receptors.

A “fusion protein” and a “fusion polypeptide” refer to a polypeptidehaving two portions covalently linked together, where each of theportions is a polypeptide having a different property. The property maybe a biological property, such as activity in vitro or in vivo. Theproperty may also be simple chemical or physical property, such asbinding to a target molecule, catalysis of a reaction, etc. The twoportions may be linked directly by a single peptide bond or through apeptide linker but are in reading frame with each other.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native-sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy-chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue. Suitable native-sequence Fc regions foruse in the antibodies of the invention include human IgG1, IgG2 (IgG2A,IgG2B), IgG3 and IgG4.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors, FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see M. Daëron, Annu.Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet,Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995).Other FcRs, including those to be identified in the future, areencompassed by the term “FcR” herein.

“Human effector cells” refer to leukocytes that express one or more FcRsand perform effector functions. In certain embodiments, the cellsexpress at least FcγRIII and perform ADCC effector function(s). Examplesof human leukocytes which mediate ADCC include peripheral bloodmononuclear cells (PBMC), natural killer (NK) cells, monocytes,cytotoxic T cells, and neutrophils. The effector cells may be isolatedfrom a native source, e.g., from blood.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

The phrase “substantially reduced,” or “substantially different,” asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with a molecule and theother associated with a reference/comparator molecule) such that one ofskill in the art would consider the difference between the two values tobe of statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, greater than about 10%, greaterthan about 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, and/or less thanabout 10% as a function of the reference/comparator value.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a nativepolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments or amino acid sequence variants of nativepolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying agonists or antagonists of apolypeptide may comprise contacting a polypeptide with a candidateagonist or antagonist molecule and measuring a detectable change in oneor more biological activities normally associated with the polypeptide.

As used herein, an “antagonist antibody” of a target molecule refers toan antibody that interferes with the normal functioning of the targetmolecule, either by decreasing transcription or translation of thetarget molecule-encoding nucleic acid, or by inhibiting or blocking thetarget molecule activity, or both. For example, an antagonist antibodyof TIGIT may be considered an antibody, or anti-TIGIT binding fragmentthereof, that interferes with the normal functioning of TIGIT, either bydecreasing transcription or translation of TIGIT-encoding nucleic acid,or by inhibiting or blocking TIGIT polypeptide activity, or both. Itwill be understood by one of ordinary skill in the art that in someinstances, an antagonist antibody of TIGIT may antagonize one TIGITactivity without affecting another TIGIT activity. For example, adesirable antagonist antibody of TIGIT for use in certain methods hereinis an antagonist antibody of TIGIT that antagonizes TIGIT activity inresponse to one of PVR interaction, PVRL3 interaction, or PVRL2interaction, e.g., without affecting or minimally affecting any of theother TIGIT interactions.

The terms “TIGIT antagonist” and “antagonist of TIGIT activity or TIGITexpression” are used interchangeably and refer to a compound thatinterferes with the normal functioning of TIGIT, either by decreasingtranscription or translation of TIGIT-encoding nucleic acid, or byinhibiting or blocking TIGIT polypeptide activity, or both. Examples ofTIGIT antagonists include, but are not limited to, antisensepolynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras,TIGIT-specific aptamers, anti-TIGIT antibodies, TIGIT-binding fragmentsof anti-TIGIT antibodies, TIGIT-binding small molecules, TIGIT-bindingpeptides, and other polypeptides that specifically bind TIGIT(including, but not limited to, TIGIT-binding fragments of one or moreTIGIT ligands, optionally fused to one or more additional domains), suchthat the interaction between the TIGIT antagonist and TIGIT results in areduction or cessation of TIGIT activity or expression. It will beunderstood by one of ordinary skill in the art that in some instances, aTIGIT antagonist may antagonize one TIGIT activity without affectinganother TIGIT activity. For example, a desirable TIGIT antagonist foruse in certain of the methods herein is a TIGIT antagonist thatantagonizes TIGIT activity in response to one of PVR interaction, PVRL3interaction, or PVRL2 interaction, e.g., without affecting or minimallyaffecting any of the other TIGIT interactions.

The terms “PVR antagonist” and “antagonist of PVR activity or PVRexpression” are used interchangeably and refer to a compound thatinterferes with the normal functioning of PVR, either by decreasingtranscription or translation of PVR-encoding nucleic acid, or byinhibiting or blocking PVR polypeptide activity, or both. Examples ofPVR antagonists include, but are not limited to, antisensepolynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras,PVR-specific aptamers, anti-PVR antibodies, PVR-binding fragments ofanti-PVR antibodies, PVR-binding small molecules, PVR-binding peptides,and other polypeptides that specifically bind PVR (including, but notlimited to, PVR-binding fragments of one or more PVR ligands, optionallyfused to one or more additional domains), such that the interactionbetween the PVR antagonist and PVR results in a reduction or cessationof PVR activity or expression. It will be understood by one of ordinaryskill in the art that in some instances, a PVR antagonist may antagonizeone PVR activity without affecting another PVR activity. For example, adesirable PVR antagonist for use in certain of the methods herein is aPVR antagonist that antagonizes PVR activity in response to TIGITinteraction without impacting the PVR-CD96 and/or PVR-CD226interactions.

The term “PD-1 axis binding antagonist” refers to a molecule thatinhibits the interaction of a PD-1 axis binding partner with either oneor more of its binding partner, so as to remove T-cell dysfunctionresulting from signaling on the PD-1 signaling axis—with a result beingto restore or enhance T-cell function (e.g., proliferation, cytokineproduction, target cell killing). As used herein, a PD-1 axis bindingantagonist includes a PD-1 binding antagonist, a PD-L1 bindingantagonist and a PD-L2 binding antagonist.

The term “PD-1 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-1 with one or more of its bindingpartners, such as PD-L1, PD-L2. In some embodiments, the PD-1 bindingantagonist is a molecule that inhibits the binding of PD-1 to one ormore of its binding partners. In a specific aspect, the PD-1 bindingantagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. Forexample, PD-1 binding antagonists include anti-PD-1 antibodies, antigenbinding fragments thereof, immunoadhesins, fusion proteins,oligopeptides and other molecules that decrease, block, inhibit,abrogate or interfere with signal transduction resulting from theinteraction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, a PD-1binding antagonist reduces the negative co-stimulatory signal mediatedby or through cell surface proteins expressed on T lymphocytes mediatedsignaling through PD-1 so as render a dysfunctional T-cell lessdysfunctional (e.g., enhancing effector responses to antigenrecognition). In some embodiments, the PD-1 binding antagonist is ananti-PD-1 antibody. In a specific aspect, a PD-1 binding antagonist isMDX-1106 (nivolumab) described herein. In another specific aspect, aPD-1 binding antagonist is MK-3475 (lambrolizumab) described herein. Inanother specific aspect, a PD-1 binding antagonist is CT-011(pidilizumab) described herein. In another specific aspect, a PD-1binding antagonist is MEDI-0680 (AMP-514) described herein. In anotherspecific aspect, a PD-1 binding antagonist is PDR001 described herein.In another specific aspect, a PD-1 binding antagonist is REGN2810described herein. In another specific aspect, a PD-1 binding antagonistis BGB-108 described herein.

The term “PD-L1 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L1 with either one or more of itsbinding partners, such as PD-1, B7-1. In some embodiments, a PD-L1binding antagonist is a molecule that inhibits the binding of PD-L1 toits binding partners. In a specific aspect, the PD-L1 binding antagonistinhibits binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, thePD-L1 binding antagonists include anti-PD-L1 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-L1 withone or more of its binding partners, such as PD-1, B7-1. In oneembodiment, a PD-L1 binding antagonist reduces the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes mediated signaling through PD-L1 so as torender a dysfunctional T-cell less dysfunctional (e.g., enhancingeffector responses to antigen recognition). In some embodiments, a PD-L1binding antagonist is an anti-PD-L1 antibody. In a specific aspect, ananti-PD-L1 antibody is YW243.55.S70 described herein. In anotherspecific aspect, an anti-PD-L1 antibody is MDX-1105 described herein. Instill another specific aspect, an anti-PD-L1 antibody is MPDL3280A(atezolizumab) described herein. In still another specific aspect, ananti-PD-L1 antibody is MED14736 (durvalumab) described herein. In stillanother specific aspect, an anti-PD-L1 antibody is YW243.55.S70described herein. In still another specific aspect, an anti-PD-L1antibody is MSB0010718C (avelumab) described herein.

The term “PD-L2 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L2 with either one or more of itsbinding partners, such as PD-1. In some embodiments, a PD-L2 bindingantagonist is a molecule that inhibits the binding of PD-L2 to one ormore of its binding partners. In a specific aspect, the PD-L2 bindingantagonist inhibits binding of PD-L2 to PD-1. In some embodiments, thePD-L2 antagonists include anti-PD-L2 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-L2 witheither one or more of its binding partners, such as PD-1. In oneembodiment, a PD-L2 binding antagonist reduces the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes mediated signaling through PD-L2 so as rendera dysfunctional T-cell less dysfunctional (e.g., enhancing effectorresponses to antigen recognition). In some embodiments, a PD-L2 bindingantagonist is an immunoadhesin.

The term “OX40,” as used herein, refers to any native OX40 from anyvertebrate source, including mammals such as primates (e.g., humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed OX40 as well as any form of OX40that results from processing in the cell. The term also encompassesnaturally occurring variants of OX40, for example, splice variants orallelic variants. The amino acid sequence of an exemplary human OX40 isset forth in SEQ ID NO: 354.

“OX40 activation” refers to activation of the OX40 receptor. Generally,OX40 activation results in signal transduction.

The term “aptamer” refers to a nucleic acid molecule that is capable ofbinding to a target molecule, such as a polypeptide. For example, anaptamer of the invention can specifically bind to a TIGIT polypeptide,or to a molecule in a signaling pathway that modulates the expression ofTIGIT. The generation and therapeutic use of aptamers are wellestablished in the art. See, for example, U.S. Pat. No. 5,475,096, andthe therapeutic efficacy of MACUGEN® (Eyetech, N.Y.) for treatingage-related macular degeneration.

The term “dysfunction,” in the context of immune dysfunction, refers toa state of reduced immune responsiveness to antigenic stimulation.

The term “dysfunctional,” as used herein, also includes refractory orunresponsive to antigen recognition, specifically, impaired capacity totranslate antigen recognition into downstream T-cell effector functions,such as proliferation, cytokine production (e.g., gamma interferon)and/or target cell killing.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted immunoglobulin bound onto Fcreceptors (FcRs) present on certain cytotoxic cells (e.g., NK cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The primary cells for mediatingADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI,FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarizedin Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92(1991). To assess ADCC activity of a molecule of interest, an in vitroADCC assay, such as that described in U.S. Pat. No. 5,500,362 or5,821,337 or U.S. Pat. No. 6,737,056 (Presta), may be performed. Usefuleffector cells for such assays include PBMC and NK cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in an animal model such as that disclosed inClynes et al. PNAS (USA) 95:652-656 (1998). An exemplary assay forassessing ADCC activity is provided in the examples herein.

The term “anergy” refers to the state of unresponsiveness to antigenstimulation resulting from incomplete or insufficient signals deliveredthrough the T-cell receptor (e.g., increase in intracellular Ca²⁺ in theabsence of ras-activation). T cell anergy can also result uponstimulation with antigen in the absence of co-stimulation, resulting inthe cell becoming refractory to subsequent activation by the antigeneven in the context of costimulation. The unresponsive state can oftenbe overridden by the presence of interleukin-2 (IL-2). Anergic T-cellsdo not undergo clonal expansion and/or acquire effector functions.

“Enhancing T cell function” means to induce, cause or stimulate aneffector or memory T cell to have a renewed, sustained or amplifiedbiological function. Examples of enhancing T-cell function include:increased secretion of γ-interferon from CD8⁺ effector T cells,increased secretion of γ-interferon from CD4+ memory and/or effectorT-cells, increased proliferation of CD4+ effector and/or memory T cells,increased proliferation of CD8+ effector T-cells, increased antigenresponsiveness (e.g., clearance), relative to such levels before theintervention. In one embodiment, the level of enhancement is at least50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. Themanner of measuring this enhancement is known to one of ordinary skillin the art.

The term “exhaustion” refers to T cell exhaustion as a state of T celldysfunction that arises from sustained TCR signaling that occurs duringmany chronic infections and cancer. It is distinguished from anergy inthat it arises not through incomplete or deficient signaling, but fromsustained signaling. It is defined by poor effector function, sustainedexpression of inhibitory receptors and a transcriptional state distinctfrom that of functional effector or memory T cells. Exhaustion preventsoptimal control of infection and tumors. Exhaustion can result from bothextrinsic negative regulatory pathways (e.g., immunoregulatorycytokines) as well as cell intrinsic negative regulatory (costimulatory)pathways (PD-1, B7-H3, B7-H4, etc.).

“Enhancing T-cell function” means to induce, cause or stimulate a T-cellto have a sustained or amplified biological function, or renew orreactivate exhausted or inactive T-cells. Examples of enhancing T-cellfunction include: increased secretion of γ-interferon from CD8⁺ T-cells,increased proliferation, increased antigen responsiveness (e.g., viral,pathogen, or tumor clearance) relative to such levels before theintervention. In one embodiment, the level of enhancement is as least50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. Themanner of measuring this enhancement is known to one of ordinary skillin the art.

A “T cell dysfunctional disorder” is a disorder or condition of T-cellscharacterized by decreased responsiveness to antigenic stimulation. In aparticular embodiment, a T-cell dysfunctional disorder is a disorderthat is specifically associated with inappropriate decreased signalingthrough OX40 and/or OX40L. In another embodiment, a T-cell dysfunctionaldisorder is one in which T-cells are anergic or have decreased abilityto secrete cytokines, proliferate, or execute cytolytic activity. In aspecific aspect, the decreased responsiveness results in ineffectivecontrol of a pathogen or tumor expressing an immunogen. Examples of Tcell dysfunctional disorders characterized by T-cell dysfunction includeunresolved acute infection, chronic infection, and tumor immunity.

“Tumor immunity” refers to the process in which tumors evade immunerecognition and clearance. Thus, as a therapeutic concept, tumorimmunity is “treated” when such evasion is attenuated, and the tumorsare recognized and attacked by the immune system. Examples of tumorrecognition include tumor binding, tumor shrinkage, and tumor clearance.

“Immunogenicity” refers to the ability of a particular substance toprovoke an immune response. Tumors are immunogenic and enhancing tumorimmunogenicity aids in the clearance of the tumor cells by the immuneresponse. Examples of enhancing tumor immunogenicity include but are notlimited to treatment with an OX40 binding agonist (e.g., anti-OX40agonist antibodies) and a TIGIT inhibitor (e.g., anti-TIGIT blockingantibodies).

“Sustained response” refers to the sustained effect on reducing tumorgrowth after cessation of a treatment. For example, the tumor size mayremain to be the same or smaller as compared to the size at thebeginning of the administration phase. In some embodiments, thesustained response has a duration at least the same as the treatmentduration, at least 1.5×, 2.0×, 2.5×, or 3.0× length of the treatmentduration.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol(PEG), and PLURONICS™.

A “package insert” refers to instructions customarily included incommercial packages of medicaments that contain information about theindications customarily included in commercial packages of medicamentsthat contain information about the indications, usage, dosage,administration, contraindications, other medicaments to be combined withthe packaged product, and/or warnings concerning the use of suchmedicaments.

As used herein, the term “treatment” refers to clinical interventiondesigned to alter the natural course of the individual or cell beingtreated during the course of clinical pathology. Desirable effects oftreatment include decreasing the rate of disease progression,ameliorating or palliating the disease state, and remission or improvedprognosis. For example, an individual is successfully “treated” if oneor more symptoms associated with cancer are mitigated or eliminated,including, but are not limited to, reducing the proliferation of (ordestroying) cancerous cells, decreasing symptoms resulting from thedisease, increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat thedisease, delaying the progression of the disease, and/or prolongingsurvival of individuals.

As used herein, “delaying progression of a disease” means to defer,hinder, slow, retard, stabilize, and/or postpone development of thedisease (such as cancer). This delay can be of varying lengths of time,depending on the history of the disease and/or individual being treated.As is evident to one skilled in the art, a sufficient or significantdelay can, in effect, encompass prevention, in that the individual doesnot develop the disease. For example, a late stage cancer, such asdevelopment of metastasis, may be delayed.

As used herein, the term “reducing or inhibiting cancer relapse” meansto reduce or inhibit tumor or cancer relapse or tumor or cancerprogression.

As used herein, “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Included in this definition are benign andmalignant cancers as well as dormant tumors or micrometastatses.Examples of cancer include but are not limited to, carcinoma, myeloma,lymphoma, blastoma, sarcoma, and leukemia. More particular examples ofsuch cancers include multiple myeloma (MM), squamous cell cancer, lungcancer (including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer), pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia), chronic lymphocytic leukemia (CLL), acutelymphoblastic leukemia (ALL), Hairy cell leukemia, chronic myeloblasticleukemia, and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.

The term “tumor” refers to all neoplastic cell growth and proliferation,whether malignant or benign, and all pre-cancerous and cancerous cellsand tissues. The terms “cancer,” “cancerous,” “cell proliferativedisorder,” “proliferative disorder” and “tumor” are not mutuallyexclusive as referred to herein.

As used herein, “metastasis” is meant the spread of cancer from itsprimary site to other places in the body. Cancer cells can break awayfrom a primary tumor, penetrate into lymphatic and blood vessels,circulate through the bloodstream, and grow in a distant focus(metastasize) in normal tissues elsewhere in the body. Metastasis can belocal or distant. Metastasis is a sequential process, contingent ontumor cells breaking off from the primary tumor, traveling through thebloodstream, and stopping at a distant site. At the new site, the cellsestablish a blood supply and can grow to form a life-threatening mass.Both stimulatory and inhibitory molecular pathways within the tumor cellregulate this behavior, and interactions between the tumor cell and hostcells in the distant site are also significant.

An “effective amount” is at least the minimum concentration required toeffect a measurable improvement or prevention of a particular disorder.An effective amount herein may vary according to factors such as thedisease state, age, sex, and weight of the patient, and the ability ofthe antibody to elicit a desired response in the individual. Aneffective amount is also one in which any toxic or detrimental effectsof the treatment are outweighed by the therapeutically beneficialeffects. For prophylactic use, beneficial or desired results includeresults such as eliminating or reducing the risk, lessening theseverity, or delaying the onset of the disease, including biochemical,histological and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. For therapeutic use, beneficial or desiredresults include clinical results such as decreasing one or more symptomsresulting from the disease, increasing the quality of life of thosesuffering from the disease, decreasing the dose of other medicationsrequired to treat the disease, enhancing effect of another medicationsuch as via targeting, delaying the progression of the disease, and/orprolonging survival. In the case of cancer or tumor, an effective amountof the drug may have the effect in reducing the number of cancer cells;reducing the tumor size; inhibiting (i.e., slow to some extent ordesirably stop) cancer cell infiltration into peripheral organs; inhibit(i.e., slow to some extent and desirably stop) tumor metastasis;inhibiting to some extent tumor growth; and/or relieving to some extentone or more of the symptoms associated with the disorder. An effectiveamount can be administered in one or more administrations. For purposesof this invention, an effective amount of drug, compound, orpharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective amount of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective amount” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during, or after administration of the other treatment modalityto the individual.

As used herein, “subject” or “individual” is meant a mammal, including,but not limited to, a human or non-human mammal, such as a bovine,equine, canine, ovine, or feline. Preferably, the subject is a human.Patients are also subjects herein.

“Chemotherapeutic agent” includes chemical compounds useful in thetreatment of cancer. Examples of chemotherapeutic agents includeerlotinib (TARCEVA®, Genentech/OSI Pharm.), bortezomib (VELCADE®,Millennium Pharm.), disulfiram, epigallocatechin gallate,salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol,lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca),sunitib (SUTENT®, Pfizer/Sugen), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), finasunate (VATALANIB®, Novartis),oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin,Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TYKERB®, GSK572016,Glaxo Smith Kline), Lonafamib (SCH 66336), sorafenib (NEXAVAR®, BayerLabs), gefitinib (IRESSA®, AstraZeneca), AG1478, alkylating agents suchas thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (includingtopotecan and irinotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);adrenocorticosteroids (including prednisone and prednisolone);cyproterone acetate; 5α-reductases including finasteride anddutasteride); vorinostat, romidepsin, panobinostat, valproic acid,mocetinostat dolastatin; aldesleukin, talc duocarmycin (including thesynthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; asarcodictyin; spongistatin; nitrogen mustards such as chlorambucil,chlomaphazine, chlorophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin γ1I andcalicheamicin ω1I (Angew Chem. Intl. Ed. Engl. 1994 33:183-186);dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®(doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethyl hydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL(paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® (docetaxel, doxetaxel; Sanofi-Aventis); chloranmbucil; GEMZAR®(gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; etoposide(VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE®(vinorelbine); novantrone; teniposide; edatrexate; daunomycin;aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; topoisomeraseinhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such asretinoic acid; and pharmaceutically acceptable salts, acids andderivatives of any of the above.

Chemotherapeutic agent also includes (i) anti-hormonal agents that actto regulate or inhibit hormone action on tumors such as anti-estrogensand selective estrogen receptor modulators (SERMs), including, forexample, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene,droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and FARESTON® (toremifine citrate); (ii)aromatase inhibitors that inhibit the enzyme aromatase, which regulatesestrogen production in the adrenal glands, such as, for example,4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate),AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR®(vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole;AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide and goserelin; buserelin, tripterelin,medroxyprogesterone acetate, diethylstilbestrol, premarin,fluoxymesterone, all transretionic acid, fenretinide, as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors; (v) lipid kinase inhibitors; (vi) antisenseoligonucleotides, particularly those which inhibit expression of genesin signaling pathways implicated in aberrant cell proliferation, suchas, for example, PKC-alpha, Ralf and H-Ras; (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN®, rIL-2; a topoisomerase 1 inhibitorsuch as LURTOTECAN®; ABARELIX® rmRH; and (ix) pharmaceuticallyacceptable salts, acids and derivatives of any of the above.

Chemotherapeutic agent also includes antibodies such as alemtuzumab(Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®,Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®,Genentech/Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech),trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), andthe antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).Additional humanized monoclonal antibodies with therapeutic potential asagents in combination with the compounds of the invention include:apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine,cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab,cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab,felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin,ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab,motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab,numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab,pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab,ustekinumab, visilizumab, and the anti-interleukin-12 (ABT-874/J695,Wyeth Research and Abbott Laboratories) which is a recombinantexclusively human-sequence, full-length IgG1 A antibody geneticallymodified to recognize interleukin-12 p40 protein.

Chemotherapeutic agent also includes “EGFR inhibitors,” which refers tocompounds that bind to or otherwise interact directly with EGFR andprevent or reduce its signaling activity, and is alternatively referredto as an “EGFR antagonist.” Examples of such agents include antibodiesand small molecules that bind to EGFR. Examples of antibodies which bindto EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507),MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No.4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized225 (C225 or Cetuximab; ERBUTIX®) and reshaped human 225 (H225) (see, WO96/40210, Imclone Systems Inc.); IMC-11F8, a fully human, EGFR-targetedantibody (Imclone); antibodies that bind type II mutant EGFR (U.S. Pat.No. 5,212,290); humanized and chimeric antibodies that bind EGFR asdescribed in U.S. Pat. No. 5,891,996; and human antibodies that bindEGFR, such as ABX-EGF or Panitumumab (see WO98/50433, Abgenix/Amgen);EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996));EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR thatcompetes with both EGF and TGF-alpha for EGFR binding (EMD/Merck); humanEGFR antibody, HuMax-EGFR (GenMab); fully human antibodies known asE1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6. 3 and E7.6. 3 and described inU.S. Pat. No. 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanizedmAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). Theanti-EGFR antibody may be conjugated with a cytotoxic agent, thusgenerating an immunoconjugate (see, e.g., EP659,439A2, Merck PatentGmbH). EGFR antagonists include small molecules such as compoundsdescribed in U.S. Pat. Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307,5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726,6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459,6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, aswell as the following PCT publications: WO98/14451, WO98/50038,WO99/09016, and WO99/24037. Particular small molecule EGFR antagonistsinclude OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech/OSIPharmaceuticals); PD 183805 (CI 1033, 2-propenamide,N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-,dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®)4-(3′-Chloro-4′-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline,AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline,Zeneca); BIBX-1382(N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine,Boehringer Ingelheim); PKI-166((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol);(R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine);CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide);EKB-569(N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide)(Wyeth); AG1478 (Pfizer); AG1571 (SU 5271; Pfizer); dual EGFR/HER2tyrosine kinase inhibitors such as lapatinib (TYKERB®, GSK572016 orN-[3-chloro-4-[(3fluorophenyl)methoxy]phenyl]-6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).

Chemotherapeutic agents also include “tyrosine kinase inhibitors”including the EGFR-targeted drugs noted in the preceding paragraph;small molecule HER2 tyrosine kinase inhibitor such as TAK165 availablefrom Takeda; CP-724,714, an oral selective inhibitor of the ErbB2receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such asEKB-569 (available from Wyeth) which preferentially binds EGFR butinhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016;available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinaseinhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such ascanertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisenseagent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1signaling; non-HER targeted TK inhibitors such as imatinib mesylate(GLEEVEC®, available from Glaxo SmithKline); multi-targeted tyrosinekinase inhibitors such as sunitinib (SUTENT®, available from Pfizer);VEGF receptor tyrosine kinase inhibitors such as vatalanib(PTK787/ZK222584, available from Novartis/Schering AG); MAPKextracellular regulated kinase I inhibitor CI-1040 (available fromPharmacia); quinazolines, such as PD 153035,4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines,such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines; curcumin (diferuloylmethane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containingnitrothiophene moieties; PD-0183805 (Warner-Lamber); antisense molecules(e.g. those that bind to HER-encoding nucleic acid); quinoxalines (U.S.Pat. No. 5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474(Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors suchas CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); imatinibmesylate (GLEEVEC®); PKI 166 (Novartis); GW2016 (Glaxo SmithKline);CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474(AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone),rapamycin (sirolimus, RAPAMUNE®); or as described in any of thefollowing patent publications: U.S. Pat. No. 5,804,396; WO 1999/09016(American Cyanamid); WO 1998/43960 (American Cyanamid); WO 1997/38983(Warner Lambert); WO 1999/06378 (Warner Lambert); WO 1999/06396 (WarnerLambert); WO 1996/30347 (Pfizer, Inc); WO 1996/33978 (Zeneca); WO1996/3397 (Zeneca) and WO 1996/33980 (Zeneca).

Chemotherapeutic agents also include dexamethasone, interferons,colchicine, metoprine, cyclosporine, amphotericin, metronidazole,alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide,asparaginase, BCG live, bevacuzimab, bexarotene, cladribine,clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa,elotinib, filgrastim, histrelin acetate, ibritumomab, interferonalfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna,methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin,palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim,pemetrexed disodium, plicamycin, porfimer sodium, quinacrine,rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene,tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, andpharmaceutically acceptable salts thereof.

Chemotherapeutic agents also include hydrocortisone, hydrocortisoneacetate, cortisone acetate, tixocortol pivalate, triamcinoloneacetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide,desonide, fluocinonide, fluocinolone acetonide, betamethasone,betamethasone sodium phosphate, dexamethasone, dexamethasone sodiumphosphate, fluocortolone, hydrocortisone-17-butyrate,hydrocortisone-17-valerate, aclometasone dipropionate, betamethasonevalerate, betamethasone dipropionate, prednicarbate,clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolonecaproate, fluocortolone pivalate and fluprednidene acetate; immuneselective anti-inflammatory peptides (ImSAIDs) such asphenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG)(IMULAN BioTherapeutics, LLC); anti-rheumatic drugs such asazathioprine, ciclosporin (cyclosporine A), D-penicillamine, gold salts,hydroxychloroquine, leflunomideminocycline, sulfasalazine, tumornecrosis factor alpha (TNFα) blockers such as etanercept (Enbrel),infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia),golimumab (Simponi), Interleukin 1 (IL-1) blockers such as anakinra(Kineret), T cell costimulation blockers such as abatacept (Orencia),Interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA®);Interleukin 13 (IL-13) blockers such as lebrikizumab; Interferon alpha(IFN) blockers such as Rontalizumab; Beta 7 integrin blockers such asrhuMAb Beta7; IgE pathway blockers such as Anti-M1 prime; Secretedhomotrimeric LTa3 and membrane bound heterotrimer LTa1/β2 blockers suchas Anti-lymphotoxin alpha (LTa); radioactive isotopes (e.g., At211,I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactiveisotopes of Lu); miscellaneous investigational agents such asthioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyl transferaseinhibitors (L-739749, L-744832); polyphenols such as quercetin,resveratrol, piceatannol, epigallocatechine gallate, theaflavins,flavanols, procyanidins, betulinic acid and derivatives thereof;autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin);podophyllotoxin; tegafur (UFTORAL®); bexarotene (TARGRETIN®);bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®),alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), orrisedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R);vaccines such as THERATOPE® vaccine; perifosine, COX-2 inhibitor (e.g.celecoxib or etoricoxib), proteosome inhibitor (e.g. PS341); CCI-779;tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such asoblimersen sodium (GENASENSE®); pixantrone; farnesyltransferaseinhibitors such as lonafarnib (SCH 6636, SARASAR™); and pharmaceuticallyacceptable salts, acids or derivatives of any of the above; as well ascombinations of two or more of the above such as CHOP, an abbreviationfor a combined therapy of cyclophosphamide, doxorubicin, vincristine,and prednisolone; and FOLFOX, an abbreviation for a treatment regimenwith oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

Chemotherapeutic agents also include non-steroidal anti-inflammatorydrugswith analgesic, antipyretic and anti-inflammatory effects. NSAIDsinclude non-selective inhibitors of the enzyme cyclooxygenase. Specificexamples of NSAIDs include aspirin, propionic acid derivatives such asibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen,acetic acid derivatives such as indomethacin, sulindac, etodolac,diclofenac, enolic acid derivatives such as piroxicam, meloxicam,tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivativessuch as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamicacid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib,parecoxib, rofecoxib, rofecoxib, and valdecoxib. NSAIDs can be indicatedfor the symptomatic relief of conditions such as rheumatoid arthritis,osteoarthritis, inflammatory arthropathies, ankylosing spondylitis,psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhoea,metastatic bone pain, headache and migraine, postoperative pain,mild-to-moderate pain due to inflammation and tissue injury, pyrexia,ileus, and renal colic.

As used herein, the term “cytokine” refers generically to proteinsreleased by one cell population that act on another cell asintercellular mediators or have an autocrine effect on the cellsproducing the proteins. Examples of such cytokines include lymphokines,monokines; interleukins (“ILs”) such as IL-1, IL-1α, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL10, IL-11, IL-12, IL-13, IL-15,IL-17A-F, IL-18 to IL-29 (such as IL-23), IL-31, including PROLEUKIN®rIL-2; a tumor-necrosis factor such as TNF-α or TNF-β, TGF-β1-3; andother polypeptide factors including leukemia inhibitory factor (“LIF”),ciliary neurotrophic factor (“CNTF”), CNTF-like cytokine (“CLC”),cardiotrophin (“CT”), and kit ligand (“KL”).

As used herein, the term “chemokine” refers to soluble factors (e.g.,cytokines) that have the ability to selectively induce chemotaxis andactivation of leukocytes. They also trigger processes of angiogenesis,inflammation, wound healing, and tumorigenesis. Example chemokinesinclude IL-8, a human homolog of murine keratinocyte chemoattractant(KC).

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

As used herein, “administering” is meant a method of giving a dosage ofa compound (e.g., an anti-TIGIT antibody of the invention or a nucleicacid encoding an anti-TIGIT antibody of the invention) or a composition(e.g., a pharmaceutical composition, e.g., a pharmaceutical compositionincluding an anti-TIGIT antibody of the invention) to a subject. Thecompositions utilized in the methods described herein can beadministered, for example, intravenously, intramuscularly,intravitreally (e.g., by intravitreal injection), by eye drop,intradermally, percutaneously, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intrathecally, intranasally,intravaginally, intrarectally, topically, intratumorally, peritoneally,subcutaneously, subconjunctivally, intravesicularly, mucosally,intrapericardially, intraumbilically, intraocularly, intraorbitally,orally, topically, transdermally, by inhalation, by injection, byimplantation, by infusion, by continuous infusion, by localizedperfusion bathing target cells directly, by catheter, by lavage, incremes, or in lipid compositions. The compositions utilized in themethods described herein can also be administered systemically orlocally. The method of administration can vary depending on variousfactors (e.g., the compound or composition being administered and theseverity of the condition, disease, or disorder being treated).

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “recombinantvectors” or “expression vectors”). In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” may be usedinterchangeably. The term “about” as used herein refers to the usualerror range for the respective value readily known to the skilled personin this technical field. Reference to “about” a value or parameterherein includes (and describes) embodiments that are directed to thatvalue or parameter per se.

III. Exemplary Anti-TIGIT Antibodies

The invention provides anti-TIGIT antibodies useful for treating ordelaying the progression of cancer or an immune-related disease (e.g., aT cell dysfunctional disorder) in a subject (e.g., a human).

In one example, the anti-TIGIT antibodies bind to an epitope on humanTIGIT including one or more amino acid residues (e.g., 1, 2, or 3 aminoacid residues) selected from the group consisting of Ser78, Ser80, andLys82 of human TIGIT. For example, in some instances, the anti-TIGITantibodies bind to an epitope on human TIGIT including amino acidresidues Ser80 and Lys82. In some instances, the anti-TIGIT antibodiesbind to an epitope on human TIGIT including amino acid residues Ser78and Ser80. In some instances, the anti-TIGIT antibodies bind to anepitope on human TIGIT including amino acid residues Ser78 and Lys82. Insome instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT including amino acid residues Ser78, Ser80, and Lys82.

In some instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT that includes, in addition to Ser78, Ser80, and/or Lys82, residueAla67 of human TIGIT. In some instances, the anti-TIGIT antibodies bindto an epitope on human TIGIT including Ser78 and Ala67; Ser80 and Ala67;Lys82 and Ala67; Ser78, Ser80, and Ala67; Ser78, Lys82, and Ala67;Ser80, Lys82, and Ala67; or Ser78, Ser80, Lys82, and Ala67.

In some instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT that includes, in addition to Ser78, Ser80, and/or Lys82, one ormore additional amino acid residues (e.g., 1, 2, or 3 amino acidresidues) selected from the group consisting of Glu60, Leu65, and Ile68of human TIGIT. In some instances, the anti-TIGIT antibodies bind to anepitope on human TIGIT that includes Ser78 and Glu60; Ser80 and Glu60;Lys82 and Glu60; Ser78 and Leu65; Ser80 and Leu65; Lys82 and Leu65;Ser78 and Ile68; Ser80 and Ile68; Lys82 and Ile68. In some instances,the anti-TIGIT antibodies bind to an epitope on human TIGIT thatincludes Ser78, Ser80, and Glu60; Ser78, Ser80, and Leu65; Ser78, Ser80,and Ile68; Ser80, Lys82, and Glu60; Ser80, Lys82, and Leu65; Ser80,Lys82, and Ile68; Ser78, Lys82, and Glu60; Ser78, Lys82, and Leu65;Ser78, Lys82, and Ile68. In some instances, the anti-TIGIT antibodiesbind to an epitope on human TIGIT that includes Ser78, Ser80, Lys82, andGlu60; Ser78, Ser80, Lys82, and Leu65; Ser78, Ser80, Lys82, and Ile68.In some instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT that includes Glu60, Leu65, and Ser78; Glu60, Leu65, and Ser80;Glu60, Leu65, and Lys82; Leu65, Ile68, and Ser78; Leu65, Ile68, andSer80; Leu65, Ile68, and Lys82; Glu60, Ile68, and Ser78; Glu60, Ile68,and Ser80; Glu60, Ile68, and Lys82. In some instances, the anti-TIGITantibodies bind to an epitope on human TIGIT that includes Glu60, Leu65,Ile68, and Ser78; Glu60, Leu65, Ile68, and Ser80; Glu60, Leu65, Ile68,and Lys82. In some instances, the anti-TIGIT antibodies bind to anepitope on human TIGIT that includes Glu60, Leu65, Ser78, and Ser80;Glu60, Leu65, Ser78, and Lys82; Glu60, Leu65, Ser80, and Lys82; Leu65,Ile68, Ser78, and Ser80; Leu65, Ile68, Ser78, and Lys82; Leu65, Ile68,Ser80, and Lys82; Leu65, Glu60, Ser78, and Ser80; Leu65, Glu60, Ser78,and Lys82; Leu65, Glu60, Ser80, and Lys82. In some instances, theanti-TIGIT antibodies bind to an epitope on human TIGIT that includesGlu60, Leu65, Ser78, Ser80, and Lys82; Leu65, Ile68, Ser78, Ser80, andLys82; Glu60, Leu65, Ser78, Ser80, and Lys82. In some instances, theanti-TIGIT antibodies bind to an epitope on human TIGIT that includesSer78, Ser80, Glu60, Leu65, and Ile68; Ser78, Lys82, Glu60, Leu65, andIle68; Ser80, and Lys82, Glu60, Leu65, and Ile68. In some instances, theanti-TIGIT antibodies bind to an epitope on human TIGIT that includesSer78, Ser80, Lys82, Glu60, Leu65, and Ile68.

In some instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT that includes, in addition to Ser78, Ser80, and Lys82, one or moreadditional amino acid residues (e.g., 1, 2, 3, or 4 amino acid residues)selected from the group consisting of Gln56, Asn70, Leu73, and His111 ofhuman TIGIT. In some instances, the anti-TIGIT antibodies bind to anepitope on human TIGIT that includes Ser78, Ser80, Lys82, and Gln56;Ser78, Ser80, Lys82, and Asn70; Ser78, Ser80, Lys82, and Leu73; Ser78,Ser80, Lys82, and His111. In some instances, the anti-TIGIT antibodiesbind to an epitope on human TIGIT that includes Ser78, Ser80, Lys82,Gln56, and Asn70; Ser78, Ser80, Lys82, Gln56, and Leu73; Ser78, Ser80,Lys82, Gln56, and His111; Ser78, Ser80, Lys82, Asn70, and Leu73; Ser78,Ser80, Lys82, Asn70, and His111; Ser78, Ser80, Lys82, Leu73, and His111.In some instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT that includes Ser78, Ser80, Lys82, Gln56, Asn70, and Leu73; Ser78,Ser80, Lys82, Gln56, Asn70, and His111; Ser78, Ser80, Lys82, Gln56,Leu73, and His111; Ser78, Ser80, Lys82, Asn70, Leu73, and His111. Insome instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT that includes Ser78, Ser80, Lys82, Gln56, Asn70, Leu73, andHis111. In some instances, the anti-TIGIT antibodies bind to an epitopeon human TIGIT that includes, in addition to Ser78, Ser80, and/or Lys82,one or more additional amino acid residues (e.g., 1, 2, or 3 amino acidresidues) selected from the group consisting of Thr55, Asn58, Asp63,Gln64, His76, Ile77, and Pro79 of human TIGIT.

In some instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT that includes amino acid residues Thr55, Gln56, Asn58, Glu60,Asp63, Gln64, Leu65, Ala67, Ile68, Asn70, Leu73, His76, Ile77, Ser78,Pro79, Ser80, Lys82, and His111 of human TIGIT. In some instances, theanti-TIGIT antibodies bind to an epitope on human TIGIT that consists ofamino acid residues Thr55, Gln56, Asn58, Glu60, Asp63, Gln64, Leu65,Ala67, Ile68, Asn70, Leu73, His76, Ile77, Ser78, Pro79, Ser80, Lys82,and His111 of human TIGIT.

In another example, the anti-TIGIT antibodies bind to an epitope onhuman TIGIT including one or more amino acid residues (e.g., 1, 2, or 3amino acid residues) selected from the group consisting of Thr55, Ser80,and Lys82 of human TIGIT. For example, in some instances, the anti-TIGITantibodies bind to an epitope on human TIGIT including amino acidresidue Lys82. In some instances, the anti-TIGIT antibodies bind to anepitope on human TIGIT including amino acid residues Ser80 and Lys82. Insome instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT including amino acid residues Thr55 and Ser80. In some instances,the anti-TIGIT antibodies bind to an epitope on human TIGIT includingamino acid residues Thr55 and Lys82. In some instances, the anti-TIGITantibodies bind to an epitope on human TIGIT including amino acidresidues Thr55, Ser80, and Lys82.

In some instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT that includes, in addition to Thr55, Ser80, and/or Lys82, residueGln56 of human TIGIT. In some instances, the anti-TIGIT antibodies bindto an epitope on human TIGIT including Thr55 and Gln56; Ser80 and Gln56;Lys82 and Gln56; Thr55, Ser80, and Gln56; Thr55, Lys82, and Gln56;Ser80, Lys82, and Gln56; or Thr55, Ser80, Lys82, and Gln56.

In some instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT that includes, in addition to Thr55, Ser80, and/or Lys82, one ormore additional amino acid residues (e.g., 1, 2, or 3 amino acidresidues) selected from the group consisting of Asn58, Glu60, Ile77, andPro79 of human TIGIT. In some instances, the anti-TIGIT antibodies bindto an epitope on human TIGIT that includes Thr55 and Asn58; Thr55 andGlu60; Ser80 and Asn58; Ser80 and Glu60; Lys82 and Asn58; Lys82 andGlu60; Thr55 and Ile77; Ser80 and Ile77; Lys82 and Ile77; Thr55 andPro79; Ser80 and Pro79; Lys82 and Pro79. In some instances, theanti-TIGIT antibodies bind to an epitope on human TIGIT that includesThr55, Ser80, and Asn58; Thr55, Ser80, and Glu60; Thr55, Ser80, andIle77; Thr55, Ser80, and Pro79; Ser80, Lys82, and Asn58; Ser80, Lys82,and Glu60; Ser80, Lys82, and Ile77; Ser80, Lys82, and Pro79; Thr55,Lys82, and Asn58; Thr55, Lys82, and Glu60; Thr55, Lys82, and Ile77;Thr55, Lys82, and Pro79. In some instances, the anti-TIGIT antibodiesbind to an epitope on human TIGIT that includes Thr55, Ser80, Lys82, andAsn58; Thr55, Ser80, Lys82, and Glu60; Thr55, Ser80, Lys82, and Ile77;Thr55, Ser80, Lys82, and Pro79. In some instances, the anti-TIGITantibodies bind to an epitope on human TIGIT that includes Asn58, Ile77,and Thr55; Glu60, Ile77, and Thr55; Asn58, Ile77, and Ser80; Glu60,Ile77, and Ser80; Asn58, Ile77, and Lys82; Glu60, Ile77, and Lys82;Ile77, Pro79, and Thr55; Ile77, Pro79, and Ser80; Ile77, Pro79, andLys82; Glu60, Pro79, and Thr55; Asn58, Pro79, and Ser80; Glu60, Pro79,and Ser80; Asn58, Pro79, and Lys82; Glu60, Pro79, and Lys82. In someinstances, the anti-TIGIT antibodies bind to an epitope on human TIGITthat includes Asn58, Ile77, Pro79, and Thr55; Glu60, Ile77, Pro79, andThr55; Asn58, Ile77, Pro79, and Ser80; Glu60, Ile77, Pro79, and Ser80;Asn58, Ile77, Pro79, and Lys82; Glu60, Ile77, Pro79, and Lys82. In someinstances, the anti-TIGIT antibodies bind to an epitope on human TIGITthat includes Asn58, Ile77, Thr55, and Ser80; Glu60, Ile77, Thr55, andSer80; Asn58, Ile77, Thr55, and Lys82; Glu60, Ile77, Thr55, and Lys82;Asn58, Ile77, Ser80, and Lys82; Glu60, Ile77, Ser80, and Lys82; Ile77,Pro79, Thr55, and Ser80; Ile77, Pro79, Thr55, and Lys82; Ile77, Pro79,Ser80, and Lys82; Ile77, Asn58, Thr55, and Ser80; Ile77, Glu60, Thr55,and Ser80; Ile77, Asn58, Thr55, and Lys82; Ile77, Asn58, Thr55, andLys82; Ile77, Glu60, Thr55, and Lys82; Ile77, Asn58, Ser80, and Lys82;Ile77, Glu60, Ser80, and Lys82. In some instances, the anti-TIGITantibodies bind to an epitope on human TIGIT that includes Asn58, Ile77,Thr55, Ser80, and Lys82; Glu60, Ile77, Thr55, Ser80, and Lys82; Ile77,Pro79, Thr55, Ser80, and Lys82; Asn58, Ile77, Thr55, Ser80, and Lys82;Glu60, Ile77, Thr55, Ser80, and Lys82. In some instances, the anti-TIGITantibodies bind to an epitope on human TIGIT that includes Thr55, Ser80,Asn58, Ile77, and Pro79; Thr55, Ser80, Glu60, Ile77, and Pro79; Thr55,Lys82, Asn58, Ile77, and Pro79; Thr55, Lys82, Glu60, Ile77, and Pro79;Ser80, Lys82, Asn58, Ile77, and Pro79; and Ser80, Lys82, Glu60, Ile77,and Pro79. In some instances, the anti-TIGIT antibodies bind to anepitope on human TIGIT that includes Thr55, Ser80, Lys82, Glu60, Ile77,and Pro79. In some instances, the anti-TIGIT antibodies bind to anepitope on human TIGIT that includes Thr55, Ser80, Lys82, Asn58, Ile77,and Pro79. In some instances, the anti-TIGIT antibodies bind to anepitope on human TIGIT that includes Thr55, Asn58, Glu60, Ile77, Pro79,Ser80, and Lys82.

In some instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT that includes, in addition to Thr55, Ser80, and Lys82, one or moreadditional amino acid residues (e.g., 1, 2, 3, 4, 5, or 6 amino acidresidues) selected from the group consisting of Leu65, Ile68, Leu73,His76, Ser78, and His111 of further comprises amino acid residues Leu65,Ile68, Leu73, His76, Ser78, and His111 of human TIGIT.

In some instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT that includes amino acid residues Thr55, Gln56, Asn58, Glu60,Leu65, Ile68, Leu73, His76, Ile77, Ser78, Pro79, Ser80, Lys82, andHis111 of human TIGIT. In some instances, the anti-TIGIT antibodies bindto an epitope on human TIGIT that consists of amino acid residues Thr55,Gln56, Asn58, Glu60, Leu65, Ile68, Leu73, His76, Ile77, Ser78, Pro79,Ser80, Lys82, and His111 of human TIGIT.

In some instances, any of the anti-TIGIT antibodies described above mayinclude a paratope that includes one or more amino acid residues (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues) selectedfrom the group consisting of heavy chain variable region amino acidresidues Asn32, Tyr52, Arg52b, Phe53, Lys54, Tyr56, Asp58, Tyr99,Asp100, Leu100a, Leu100b, and Ala100c and/or one or more amino acidresidues (e.g., 1, 2, 3, 4, or 5 amino acid residues) selected from thegroup consisting of light chain variable region amino acid residuesTyr27d, Tyr92, Ser93, Thr94, and Phe96.

In some instances, the anti-TIGIT antibodies may include a paratope thatconsists of heavy chain variable region amino acid residues Asn32,Tyr52, Arg52b, Phe53, Lys54, Tyr56, Asp58, Tyr99, Asp100, Leu100a,Leu100b, and Ala100c and light chain variable region amino acid residuesTyr27d, Tyr92, Ser93, Thr94, and Phe96.

In certain embodiments, the anti-TIGIT antibody of the invention makesunique contacts with amino acids of human TIGIT at a distance of 4.5Angstroms, 3.7 Angstroms, 3.5 Angstroms, 3.25 Angstroms, 3.00 Angstroms,2.75 Angstroms, or less. In certain embodiments, an antibody is providedthat binds to an epitope consisting of one, two, three, four, or fiveamino acids of human TIGIT at a distance of 4.5 Angstroms, 3.7Angstroms, 3.5 Angstroms, 3.25 Angstroms, 3.00 Angstroms, 2.75 Angstromsor less. In one embodiment, the anti-TIGIT antibody of the inventionmakes unique contacts with amino acids of human TIGIT at a distance of3.7 Angstroms or less. In certain embodiments, an antibody is providedthat binds to an epitope consisting of one, two, three, four, or fiveamino acids of human TIGIT at a distance of 3.7 Angstroms or less.

In some instances, any of the above anti-TIGIT antibodies includes atleast one, two, three, four, five, or six HVRs selected from (a) anHVR-H1 comprising the amino acid sequence of SNSAAWN (SEQ ID NO: 1); (b)an HVR-H2 comprising the amino acid sequence of KTYYRFKWYSDYAVSVKG (SEQID NO: 2); (c) an HVR-H3 comprising the amino acid sequence ofESTTYDLLAGPFDY (SEQ ID NO: 3); (d) an HVR-L1 comprising the amino acidsequence of KSSQTVLYSSNNKKYLA (SEQ ID NO: 4); (e) an HVR-L2 comprisingthe amino acid sequence of WASTRES (SEQ ID NO: 5); and/or (f) an HVR-L3comprising the amino acid sequence of QQYYSTPFT (SEQ ID NO: 6), or acombination of one or more of the above HVRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 1-6. In some instances, any of the above anti-TIGIT antibodiesincludes (a) an HVR-H1 comprising the amino acid sequence of SNSAAWN(SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequence ofKTYYRFKWYSDYAVSVKG (SEQ ID NO: 2); (c) an HVR-H3 comprising the aminoacid sequence of ESTTYDLLAGPFDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of KSSQTVLYSSNNKKYLA (SEQ ID NO: 4); (e) anHVR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 5); and(f) an HVR-L3 comprising the amino acid sequence of QQYYSTPFT (SEQ IDNO: 6), such as possessed by the anti-TIGIT antibody 4.1D3 andderivatives thereof (e.g., 4.1D3.Q1E). In some instances, the anti-TIGITantibody may have a VH domain comprising an amino acid sequence havingat least at least 90% sequence identity (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequenceof, SEQ ID NO: 34 or 35 and/or a VL domain comprising an amino acidsequence having at least 90% sequence identity (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, SEQ ID NO: 36. In some instances, the anti-TIGIT antibodymay have a VH domain comprising an amino acid sequence having at leastat least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ IDNO: 34 and/or a VL domain comprising an amino acid sequence having atleast 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ IDNO: 36. In a particular instance, the anti-TIGIT antibody can be4.1D3.Q1E, or a derivative or clonal relative thereof. In someinstances, the anti-TIGIT antibody may have a VH domain comprising anamino acid sequence having at least at least 90% sequence identity(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity) to, or the sequence of, SEQ ID NO: 35 and/or a VL domaincomprising an amino acid sequence having at least 90% sequence identity(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity) to, or the sequence of, SEQ ID NO: 36. In a particularinstance, the anti-TIGIT antibody can be 4.1D3, or a derivative orclonal relative thereof.

In some instances, the antibody further comprises at least one, two,three, or four of the following light chain variable region frameworkregions (FRs): an FR-L1 comprising the amino acid sequence ofDIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 7); an FR-L2 comprising the aminoacid sequence of WYQQKPGQPPNLLIY (SEQ ID NO: 8); an FR-L3 comprising theamino acid sequence of GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 9);and/or an FR-L4 comprising the amino acid sequence of FGPGTKVEIK (SEQ IDNO: 10), or a combination of one or more of the above FRs and one ormore variants thereof having at least about 90% sequence identity (e.g.,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any oneof SEQ ID NOs: 7-10. In some instances, for example, the antibodyfurther comprises an FR-L1 comprising the amino acid sequence ofDIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 7); an FR-L2 comprising the aminoacid sequence of WYQQKPGQPPNLLIY (SEQ ID NO: 8); an FR-L3 comprising theamino acid sequence of GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 9);and an FR-L4 comprising the amino acid sequence of FGPGTKVEIK (SEQ IDNO: 10), such as possessed by the anti-TIGIT antibodies 4.1D3.Q1E and4.1D3.

In some instances, the antibody further comprises at least one, two,three, or four of the following heavy chain variable region FRs: anFR-H1 comprising the amino acid sequence ofX₁VQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 11), wherein X₁ is Q or E;an FR-H2 comprising the amino acid sequence of WIRQSPSRGLEWLG (SEQ IDNO: 12); an FR-H3 comprising the amino acid sequence ofRITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 13); and/or an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 14), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 11-14. The anti-TIGIT antibody may further include, for example, atleast one, two, three, or four of the following heavy chain variableregion FRs: an FR-H1 comprising the amino acid sequence ofEVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 15); an FR-H2 comprising theamino acid sequence of WIRQSPSRGLEWLG (SEQ ID NO: 12); an FR-H3comprising the amino acid sequence of RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR(SEQ ID NO: 13); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 14), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 12-15. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 15); an FR-H2 comprisingthe amino acid sequence of WIRQSPSRGLEWLG (SEQ ID NO: 12); an FR-H3comprising the amino acid sequence of RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR(SEQ ID NO: 13); and an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 14), such as possessed by the 4.1D3.Q1Eantibody. In another instance, for example, the anti-TIGIT antibody mayfurther include at least one, two, three, or four of the following heavychain variable region FRs: an FR-H1 comprising the amino acid sequenceof QVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 16); an FR-H2 comprisingthe amino acid sequence of WIRQSPSRGLEWLG (SEQ ID NO: 12); an FR-H3comprising the amino acid sequence of RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR(SEQ ID NO: 13); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 14), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 12-14 and 16. In some instances,the anti-TIGIT antibody includes an FR-H1 comprising the amino acidsequence of QVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 16); an FR-H2comprising the amino acid sequence of WIRQSPSRGLEWLG (SEQ ID NO: 12); anFR-H3 comprising the amino acid sequence ofRITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 13); and an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 14), suchas possessed by the 4.1D3 antibody.

In another example, the invention provides anti-TIGIT antibodies thatbind to an epitope on human TIGIT including one or more amino acidresidues (e.g., 1, 2, or 3 amino acid residues) selected from the groupconsisting of Gln53, His111, and Tyr113 of human TIGIT. For example, insome instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT including amino acid residues His111 and Tyr113. In someinstances, the anti-TIGIT antibodies bind to an epitope on human TIGITincluding amino acid residues Gln53 and His111. In some instances, theanti-TIGIT antibodies bind to an epitope on human TIGIT including aminoacid residues Gln53 and Tyr113. In some instances, the anti-TIGITantibodies bind to an epitope on human TIGIT including amino acidresidues Gln53, His111, and Tyr113.

In some instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT that includes, in addition to Gln53, His111, and/or Tyr113,residue Gln56 of human TIGIT. In some instances, the anti-TIGITantibodies bind to an epitope on human TIGIT including Gln53 and Gln56;His111 and Gln56; Tyr113 and Gln56; Gln53, His111, and Gln56; Gln53,Tyr113, and Gln56; His111, Tyr113, and Gln56; or Gln53, His111, Tyr113,and Gln56.

In some instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT that includes, in addition Gln53, His111, Tyr113, and/or Gln56,and one or more additional amino acid residues (e.g., 1, 2, 3, 4, 5, or6 amino acid residues) selected from the group consisting of Glu60,Leu65, Ile68, Asn70, Leu73, and His76 of human TIGIT. For example, insome instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT including Gln53, His111, Tyr113, Gln56, and Glu60; Gln53, His111,Tyr113, Gln56, and Leu65; Gln53, His111, Tyr113, Gln56, and Ile68;Gln53, His111, Tyr113, Gln56, and Asn70; Gln53, His111, Tyr113, Gln56,and Leu73; Gln53, His111, Tyr113, Gln56, and His76. In some instances,the anti-TIGIT antibodies bind to an epitope on human TIGIT includingGln53, His111, Tyr113, Gln56, Glu60, and Leu65; Gln53, His111, Tyr113,Gln56, Glu60, and Ile68; Gln53, His111, Tyr113, Gln56, Glu60, and Asn70;Gln53, His111, Tyr113, Gln56, Glu60, and Leu73; Gln53, His111, Tyr113,Gln56, Glu60, and His76. In some instances, the anti-TIGIT antibodiesbind to an epitope on human TIGIT including Gln53, His111, Tyr113,Gln56, Glu60, Leu65, and Ile68; Gln53, His111, Tyr113, Gln56, Glu60,Leu65, and Asn70; Gln53, His111, Tyr113, Gln56, Glu60, Leu65, and Leu73;Gln53, His111, Tyr113, Gln56, Glu60, Leu65, and His76. In someinstances, the anti-TIGIT antibodies bind to an epitope on human TIGITincluding Gln53, His111, Tyr113, Gln56, Glu60, Ile68, and Asn70; Gln53,His111, Tyr113, Gln56, Glu60, Ile68, and Leu73; Gln53, His111, Tyr113,Gln56, Glu60, Ile68, and His76. In some instances, the anti-TIGITantibodies bind to an epitope on human TIGIT including Gln53, His111,Tyr113, Gln56, Glu60, Asn70, and Leu73; Gln53, His111, Tyr113, Gln56,Glu60, Asn70, and His76. In some instances, the anti-TIGIT antibodiesbind to an epitope on human TIGIT including Gln53, His111, Tyr113,Gln56, Glu60, Leu73, and His76. In some instances, the anti-TIGITantibodies bind to an epitope on human TIGIT including Gln53, His111,Tyr113, Gln56, Glu60, Leu65, Ile68, and Asn70; Gln53, His111, Tyr113,Gln56, Glu60, Leu65, Ile68, and Leu73; Gln53, His111, Tyr113, Gln56,Glu60, Leu65, Ile68, and His76. In some instances, the anti-TIGITantibodies bind to an epitope on human TIGIT including Gln53, His111,Tyr113, Gln56, Glu60, Ile68, Asn70, and Leu73; Gln53, His111, Tyr113,Gln56, Glu60, Ile68, Asn70, and His76. In some instances, the anti-TIGITantibodies bind to an epitope on human TIGIT including Gln53, His111,Tyr113, Gln56, Glu60, Leu65, Ile68, Asn70, and Leu73; Gln53, His111,Tyr113, Gln56, Glu60, Leu65, Ile68, Asn70, and His76; Gln53, His111,Tyr113, Gln56, Glu60, Ile68, Asn70, Leu73 and His76. In some instances,the anti-TIGIT antibodies bind to an epitope on human TIGIT includingGln53, His111, Tyr113, Gln56, Glu60, Leu65, Ile68, Asn70, Leu73, andHis76.

In some instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT including Gln53, His111, Tyr113, Gln56, Leu65, and Ile68; Gln53,His111, Tyr113, Gln56, Leu65, and Asn70; Gln53, His111, Tyr113, Gln56,Leu65, and Leu73; Gln53, His111, Tyr113, Gln56, Leu65, and His76. Insome instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT including Gln53, His111, Tyr113, Gln56, Leu65, Ile68, and Asn70;Gln53, His111, Tyr113, Gln56, Leu65, Ile68, and Leu73; Gln53, His111,Tyr113, Gln56, Leu65, Ile68, and Asn70; Gln53, His111, Tyr113, Gln56,Leu65, Ile68, and His76. In some instances, the anti-TIGIT antibodiesbind to an epitope on human TIGIT including Gln53, His111, Tyr113,Gln56, Leu65, Asn70, and Leu73; Gln53, His111, Tyr113, Gln56, Leu65,Asn70, and His76. In some instances, the anti-TIGIT antibodies bind toan epitope on human TIGIT including Gln53, His111, Tyr113, Gln56, Leu65,Leu73, and His76. In some instances, the anti-TIGIT antibodies bind toan epitope on human TIGIT including Gln53, His111, Tyr113, Gln56, Leu65,Ile68, Asn70, and Leu73; Gln53, His111, Tyr113, Gln56, Leu65, Ile68,Asn70, and His76. In some instances, the anti-TIGIT antibodies bind toan epitope on human TIGIT including Gln53, His111, Tyr113, Gln56, Leu65,Asn70, Leu73, and His76. In some instances, the anti-TIGIT antibodiesbind to an epitope on human TIGIT including Gln53, His111, Tyr113,Gln56, Leu65, Ile68, Asn70, Leu73, and His76.

In some instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT including Gln53, His111, Tyr113, Gln56, Ile68, and Asn70; Gln53,His111, Tyr113, Gln56, Ile68, and Leu73; Gln53, His111, Tyr113, Gln56,Ile68, and His76. In some instances, the anti-TIGIT antibodies bind toan epitope on human TIGIT including Gln53, His111, Tyr113, Gln56, Ile68,Asn70, and Leu73; Gln53, His111, Tyr113, Gln56, Ile68, Asn70, and His76;Gln53, His111, Tyr113, Gln56, Ile68, Leu73, and His76. In someinstances, the anti-TIGIT antibodies bind to an epitope on human TIGITincluding Gln53, His111, Tyr113, Gln56, Ile68, Asn70, Leu73, and His76.

In some instances, the anti-TIGIT antibodies bind to an epitope on humanTIGIT including Gln53, His111, Tyr113, Gln56, Asn70, and Leu73; Gln53,His111, Tyr113, Gln56, Asn70, and His76. In some instances, theanti-TIGIT antibodies bind to an epitope on human TIGIT including Gln53,His111, Tyr113, Gln56, Asn70, Leu73, and His76. In some instances, theanti-TIGIT antibodies bind to an epitope on human TIGIT including Gln53,His111, Tyr113, Gln56, Leu73, and His76.

In some instances, any of the anti-TIGIT antibodies of the precedingexample includes at least one, two, three, four, five, or six HVRsselected from (a) an HVR-H1 comprising the amino acid sequence of SYPMN(SEQ ID NO: 17); (b) an HVR-H2 comprising the amino acid sequence ofWINTNTGNPTYVQGFTG (SEQ ID NO: 18); (c) an HVR-H3 comprising the aminoacid sequence of TGGHTYDSYAFDV (SEQ ID NO: 19); (d) an HVR-L1 comprisingthe amino acid sequence of RASQVISSSLA (SEQ ID NO: 20); (e) an HVR-L2comprising the amino acid sequence of AASTLQS (SEQ ID NO: 21); and/or(f) an HVR-L3 comprising the amino acid sequence of QHLHGYPX₁N (SEQ IDNO: 22), wherein X₁ is C or S, or a combination of one or more of theabove HVRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 17-22. In some instances, any ofthe above anti-TIGIT antibodies of the preceding example may include,for example, (a) an HVR-H1 comprising the amino acid sequence of SYPMN(SEQ ID NO: 17); (b) an HVR-H2 comprising the amino acid sequence ofWINTNTGNPTYVQGFTG (SEQ ID NO: 18); (c) an HVR-H3 comprising the aminoacid sequence of TGGHTYDSYAFDV (SEQ ID NO: 19); (d) an HVR-L1 comprisingthe amino acid sequence of RASQVISSSLA (SEQ ID NO: 20); (e) an HVR-L2comprising the amino acid sequence of AASTLQS (SEQ ID NO: 21); and (f)an HVR-L3 comprising the amino acid sequence of QHLHGYPX₁N (SEQ ID NO:22), such as possessed by the anti-TIGIT antibody 7.4A3 and derivativesthereof (e.g., 7.4A3.C96S.Q1E). In some instances, the anti-TIGITantibody may include (a) an HVR-H1 comprising the amino acid sequence ofSYPMN (SEQ ID NO: 17); (b) an HVR-H2 comprising the amino acid sequenceof WINTNTGNPTYVQGFTG (SEQ ID NO: 18); (c) an HVR-H3 comprising the aminoacid sequence of TGGHTYDSYAFDV (SEQ ID NO: 19); (d) an HVR-L1 comprisingthe amino acid sequence of RASQVISSSLA (SEQ ID NO: 20); (e) an HVR-L2comprising the amino acid sequence of AASTLQS (SEQ ID NO: 21); and (f)an HVR-L3 comprising the amino acid sequence of QHLHGYPSN (SEQ ID NO:23), such as possessed by the anti-TIGIT antibody 7.4A3.C96S.Q1E. Inother instances, the anti-TIGIT antibody may include (a) an HVR-H1comprising the amino acid sequence of SYPMN (SEQ ID NO: 17); (b) anHVR-H2 comprising the amino acid sequence of WINTNTGNPTYVQGFTG (SEQ IDNO: 18); (c) an HVR-H3 comprising the amino acid sequence ofTGGHTYDSYAFDV (SEQ ID NO: 19); (d) an HVR-L1 comprising the amino acidsequence of RASQVISSSLA (SEQ ID NO: 20); (e) an HVR-L2 comprising theamino acid sequence of AASTLQS (SEQ ID NO: 21); and (f) an HVR-L3comprising the amino acid sequence of QHLHGYPCN (SEQ ID NO: 28), such aspossessed by the anti-TIGIT antibody 7.4A3.

In some instances, the anti-TIGIT antibody may have a VH domaincomprising an amino acid sequence having at least at least 90% sequenceidentity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity) to, or the sequence of, SEQ ID NO: 37 and/or a VLdomain comprising an amino acid sequence having at least 90% sequenceidentity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity) to, or the sequence of, SEQ ID NO: 38. In aparticular instance, the anti-TIGIT antibody can be 7.4A3.C96S.Q1E, or aderivative or clonal relative thereof. In some instances, the anti-TIGITantibody may have a VH domain comprising an amino acid sequence havingat least at least 90% sequence identity (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequenceof, SEQ ID NO: 39 and/or a VL domain comprising an amino acid sequencehaving at least 90% sequence identity (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequenceof, SEQ ID NO: 40. In a particular instance, the anti-TIGIT antibody canbe 7.4A3, or a derivative or clonal relative thereof.

In some instances, the antibody further comprises at least one, two,three, or four of the following light chain variable region frameworkregions (FRs): an DIQLTQSPTFLSASVGDRVTITC (SEQ ID NO: 30); an FR-L2comprising the amino acid sequence of WYQQNPGKAPKLLIY (SEQ ID NO: 31);an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTEFTLTISSLQPEDFVTYYC (SEQ ID NO: 32); and/or an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 33), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 30-33, such as possessed by the anti-TIGIT antibody 7.4A3 andderivatives thereof (e.g., 7.4A3.C96S.Q1E).

In some instances, the antibody further comprises at least one, two,three, or four of the following heavy chain variable region FRs: anFR-H1 comprising the amino acid sequence ofEVQLVQSGSDLKKPGASVRVSCKASGYTFT (SEQ ID NO: 24) or the amino acidsequence of QVQLVQSGSDLKKPGASVRVSCKASGYTFT (SEQ ID NO: 29); an FR-H2comprising the amino acid sequence of WVRQAPGHGLEWMG (SEQ ID NO: 25); anFR-H3 comprising the amino acid sequence ofRFVFSLDTSVNTAYLQISSLKAEDTAVYFCAR (SEQ ID NO: 26); and/or an FR-H4comprising the amino acid sequence of WGQGTMVTVSS (SEQ ID NO: 27), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 24-27 and 29. The anti-TIGIT antibody may further include, forexample, at least one, two, three, or four of the following heavy chainvariable region FRs: an FR-H1 comprising the amino acid sequence ofEVQLVQSGSDLKKPGASVRVSCKASGYTFT (SEQ ID NO: 24); an FR-H2 comprising theamino acid sequence of WVRQAPGHGLEWMG (SEQ ID NO: 25); an FR-H3comprising the amino acid sequence of RFVFSLDTSVNTAYLQISSLKAEDTAVYFCAR(SEQ ID NO: 26); and/or an FR-H4 comprising the amino acid sequence ofWGQGTMVTVSS (SEQ ID NO: 27), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 24-27. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLVQSGSDLKKPGASVRVSCKASGYTFT (SEQ ID NO: 24); an FR-H2 comprisingthe amino acid sequence of WVRQAPGHGLEWMG (SEQ ID NO: 25); an FR-H3comprising the amino acid sequence of RFVFSLDTSVNTAYLQISSLKAEDTAVYFCAR(SEQ ID NO: 26); and an FR-H4 comprising the amino acid sequence ofWGQGTMVTVSS (SEQ ID NO: 27), such as possessed by the 7.4A3.C96S.Q1Eantibody. In another instance, for example, the anti-TIGIT antibody mayfurther include at least one, two, three, or four of the following heavychain variable region FRs: an FR-H1 comprising the amino acid sequenceof QVQLVQSGSDLKKPGASVRVSCKASGYTFT (SEQ ID NO: 29); an FR-H2 comprisingthe amino acid sequence of WVRQAPGHGLEWMG (SEQ ID NO: 25); an FR-H3comprising the amino acid sequence of RFVFSLDTSVNTAYLQISSLKAEDTAVYFCAR(SEQ ID NO: 26); and/or an FR-H4 comprising the amino acid sequence ofWGQGTMVTVSS (SEQ ID NO: 27), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 25-27 and 29. In some instances,the anti-TIGIT antibody includes an FR-H1 comprising the amino acidsequence of QVQLVQSGSDLKKPGASVRVSCKASGYTFT (SEQ ID NO: 29); an FR-H2comprising the amino acid sequence of WVRQAPGHGLEWMG (SEQ ID NO: 25); anFR-H3 comprising the amino acid sequence ofRFVFSLDTSVNTAYLQISSLKAEDTAVYFCAR (SEQ ID NO: 26); and an FR-H4comprising the amino acid sequence of WGQGTMVTVSS (SEQ ID NO: 27), suchas possessed by the 7.4A3 anti-TIGIT antibody.

In another example, the invention provides anti-TIGIT antibodies thatinclude at least one, two, three, four, five, or six HVRs selected from(a) an HVR-H1 comprising the amino acid sequence of NYPMN (SEQ ID NO:41); (b) an HVR-H2 comprising the amino acid sequence ofWINTNTGSPAYAQDFTE (SEQ ID NO: 42); (c) an HVR-H3 comprising the aminoacid sequence of TAITSVYHFDY (SEQ ID NO: 43); (d) an HVR-L1 comprisingthe amino acid sequence of RASQGISSYLA (SEQ ID NO: 44); (e) an HVR-L2comprising the amino acid sequence of GATTLQS (SEQ ID NO: 45); and/or(f) an HVR-L3 comprising the amino acid sequence of QKLNSHPX₁S (SEQ IDNO: 46), wherein X₁ is C, S, or Y, or a combination of one or more ofthe above HVRs and one or more variants thereof having at least about90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity) to any one of SEQ ID NOs: 41-46. In someinstances, any of the above anti-TIGIT antibodies of the precedingexample may include, for example, (a) an HVR-H1 comprising the aminoacid sequence of NYPMN (SEQ ID NO: 41); (b) an HVR-H2 comprising theamino acid sequence of WINTNTGSPAYAQDFTE (SEQ ID NO: 42); (c) an HVR-H3comprising the amino acid sequence of TAITSVYHFDY (SEQ ID NO: 43); (d)an HVR-L1 comprising the amino acid sequence of RASQGISSYLA (SEQ ID NO:44); (e) an HVR-L2 comprising the amino acid sequence of GATTLQS (SEQ IDNO: 45); and (f) an HVR-L3 comprising the amino acid sequence ofQKLNSHPX₁S (SEQ ID NO: 46), wherein X₁ is C, S, or Y, such as possessedby the anti-TIGIT antibody 4.1A4 and derivatives thereof (e.g.,4.1A4.C96S.Q1E and 4.1A4.C96Y.Q1E). In some instances, the anti-TIGITantibody may include (a) an HVR-H1 comprising the amino acid sequence ofNYPMN (SEQ ID NO: 41); (b) an HVR-H2 comprising the amino acid sequenceof WINTNTGSPAYAQDFTE (SEQ ID NO: 42); (c) an HVR-H3 comprising the aminoacid sequence of TAITSVYHFDY (SEQ ID NO: 43); (d) an HVR-L1 comprisingthe amino acid sequence of RASQGISSYLA (SEQ ID NO: 44); (e) an HVR-L2comprising the amino acid sequence of GATTLQS (SEQ ID NO: 45); and (f)an HVR-L3 comprising the amino acid sequence of QKLNSHPCS (SEQ ID NO:47), such as possessed by the anti-TIGIT antibody 4.1A4. In otherinstances, the anti-TIGIT antibody may include (a) an HVR-H1 comprisingthe amino acid sequence of NYPMN (SEQ ID NO: 41); (b) an HVR-H2comprising the amino acid sequence of WINTNTGSPAYAQDFTE (SEQ ID NO: 42);(c) an HVR-H3 comprising the amino acid sequence of TAITSVYHFDY (SEQ IDNO: 43); (d) an HVR-L1 comprising the amino acid sequence of RASQGISSYLA(SEQ ID NO: 44); (e) an HVR-L2 comprising the amino acid sequence ofGATTLQS (SEQ ID NO: 45); and (f) an HVR-L3 comprising the amino acidsequence of QKLNSHPSS (SEQ ID NO: 48), such as possessed by theanti-TIGIT antibody 4.1A4.C96S.Q1E. In other instances, the anti-TIGITantibody may include (a) an HVR-H1 comprising the amino acid sequence ofNYPMN (SEQ ID NO: 41); (b) an HVR-H2 comprising the amino acid sequenceof WINTNTGSPAYAQDFTE (SEQ ID NO: 42); (c) an HVR-H3 comprising the aminoacid sequence of TAITSVYHFDY (SEQ ID NO: 43); (d) an HVR-L1 comprisingthe amino acid sequence of RASQGISSYLA (SEQ ID NO: 44); (e) an HVR-L2comprising the amino acid sequence of GATTLQS (SEQ ID NO: 45); and (f)an HVR-L3 comprising the amino acid sequence of QKLNSHPYS (SEQ ID NO:49), such as possessed by the anti-TIGIT antibody 4.1A4.C96Y.Q1E.

In some instances, the antibody further comprises at least one, two,three, or four of the following light chain variable region frameworkregions (FRs): an FR-L1 comprising the amino acid sequence ofDIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 56); an FR-L2 comprising the aminoacid sequence of WYQQKPGKAPRVLIY (SEQ ID NO: 57); an FR-L3 comprisingthe amino acid sequence of GVPSRFSGSESGTEFTLTISSLQPEDLATYYC (SEQ ID NO:58); and/or an FR-L4 comprising the amino acid sequence of FGQGTKVEIK(SEQ ID NO: 59), or a combination of one or more of the above FRs andone or more variants thereof having at least about 90% sequence identity(e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) toany one of SEQ ID NOs: 56-59, such as possessed by the anti-TIGITantibody 4.1A4 and derivatives thereof (e.g., 1A4.C96S.Q1E and1A4.C96Y.Q1E).

In some instances, the antibody further comprises at least one, two,three, or four of the following heavy chain variable region FRs: anFR-H1 comprising the amino acid sequence ofX₁VQLVQSGSELKKPGASVKVSCKASGYTLT (SEQ ID NO: 50), wherein X₁ is E or Q;an FR-H2 comprising the amino acid sequence of WVRQAPGRGLEWMG (SEQ IDNO: 51); an FR-H3 comprising the amino acid sequence ofRFVFSLDTSVTTAYLQISSLKAEDTAVYYCAR (SEQ ID NO: 52); and/or an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 53), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 50-53. The anti-TIGIT antibody may further include, for example, atleast one, two, three, or four of the following heavy chain variableregion FRs: an FR-H1 comprising the amino acid sequence ofQVQLVQSGSELKKPGASVKVSCKASGYTLT (SEQ ID NO: 54); an FR-H2 comprising theamino acid sequence of WVRQAPGRGLEWMG (SEQ ID NO: 51); an FR-H3comprising the amino acid sequence of RFVFSLDTSVTTAYLQISSLKAEDTAVYYCAR(SEQ ID NO: 52); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 53), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 51-54, such as possessed by theanti-TIGIT antibody 4.1A4. In some instances, the anti-TIGIT antibodyincludes an FR-H1 comprising the amino acid sequence ofEVQLVQSGSELKKPGASVKVSCKASGYTLT (SEQ ID NO: 55); an FR-H2 comprising theamino acid sequence of WVRQAPGRGLEWMG (SEQ ID NO: 51); an FR-H3comprising the amino acid sequence of RFVFSLDTSVTTAYLQISSLKAEDTAVYYCAR(SEQ ID NO: 52); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 53), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 51-53 and 55, such as possessedby the anti-TIGIT antibody 1A4.C96S.Q1E or 1A4.C96Y.Q1E.

In another example, the invention provides anti-TIGIT antibodies thatinclude at least one, two, three, four, five, or six HVRs selected from(a) an HVR-H1 comprising the amino acid sequence of TYGMGVS (SEQ ID NO:68); (b) an HVR-H2 comprising the amino acid sequence ofSIWWNGNTYYNPSLKS (SEQ ID NO: 69); (c) an HVR-H3 comprising the aminoacid sequence of TGGAVITWFAY (SEQ ID NO: 70); (d) an HVR-L1 comprisingthe amino acid sequence of KASQSVGKNIA (SEQ ID NO: 71); (e) an HVR-L2comprising the amino acid sequence of YASNRYT (SEQ ID NO: 72); and/or(f) an HVR-L3 comprising the amino acid sequence of QHIYNSPYP (SEQ IDNO: 73), or a combination of one or more of the above HVRs and one ormore variants thereof having at least about 90% sequence identity (e.g.,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any oneof SEQ ID NOs: 68-73. In some instances, any of the above anti-TIGITantibodies of the preceding example may include, for example, (a) anHVR-H1 comprising the amino acid sequence of TYGMGVS (SEQ ID NO: 68);(b) an HVR-H2 comprising the amino acid sequence of SIWWNGNTYYNPSLKS(SEQ ID NO: 69); (c) an HVR-H3 comprising the amino acid sequence ofTGGAVITWFAY (SEQ ID NO: 70); (d) an HVR-L1 comprising the amino acidsequence of KASQSVGKNIA (SEQ ID NO: 71); (e) an HVR-L2 comprising theamino acid sequence of YASNRYT (SEQ ID NO: 72); and (f) an HVR-L3comprising the amino acid sequence of QHIYNSPYP (SEQ ID NO: 73), such aspossessed by the anti-TIGIT antibody rat6B2 and derivatives thereof(e.g., h6B2.L1H1, h6B2.L2H1, h6B2.L1H2, h6B2.L1H3, h6B2.L1H4, h6B2.L1H5,and h6B2.L2H5).

In some instances, the antibody further comprises at least one, two,three, or four of the following light chain variable region frameworkregions (FRs): an FR-L1 comprising the amino acid sequence ofX₁IX₂MTQSPX₃SX₄SX₅SVGDRVTX₆X₇C (SEQ ID NO: 88), wherein X₁ is D or N; X₂is Q or V; X₃ is K or S; X₄ is L or M; X₅ is A or I; X₆ is I or M; andX₇ is N or T; an FR-L2 comprising the amino acid sequence ofWYQQKPGX₁X₂PKLLIY (SEQ ID NO: 89), wherein X₁ is K or Q and X₂ is A orS; an FR-L3 comprising the amino acid sequence ofGVPX₁RFX₂GX₃GSGTDFTX₄TIX₅X₆X₇QX₈EDX₉AX₁₀X₁₁YC (SEQ ID NO: 90), whereinX₁ is D or S; X₂ is S or T; X₃ is G or S; X₄ is F or L; X₅ is N or S; X₆is S or T; X₇ is L or V; X₈ is A or P; X₉ is A or I; X₁₀ is F or T; andX₁₁ is F or Y; and/or an FR-L4 comprising the amino acid sequence ofFGX₁GTKX₂EIK (SEQ ID NO: 91), wherein X₁ is Q or T and X₂ is L or V, ora combination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 88-91, such as possessed by the anti-TIGIT antibody rat6B2 andderivatives thereof (e.g., h6B2.L1H1, h6B2.L2H1, h6B2.L1H2, h6B2.L1H3,h6B2.L1H4, h6B2.L1H5, and h6B2.L2H5). The anti-TIGIT antibody mayfurther include, for example, at least one, two, three, or four of thefollowing light chain variable region FRs: an FR-L1 comprising the aminoacid sequence of NIVMTQSPKSMSISVGDRVTMNC (SEQ ID NO: 92); an FR-L2comprising the amino acid sequence of WYQQKPGQSPKLLIY (SEQ ID NO: 93);an FR-L3 comprising the amino acid sequence ofGVPDRFTGGGSGTDFTLTINTVQAEDAAFFYC (SEQ ID NO: 94); and/or an FR-L4comprising the amino acid sequence of FGTGTKLEIK (SEQ ID NO: 95), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 92-65, such as possessed by the anti-TIGIT antibody rat6B2. In someinstances, the anti-TIGIT antibody includes an FR-L1 comprising theamino acid sequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 96); an FR-L2comprising the amino acid sequence of WYQQKPGKSPKLLIY (SEQ ID NO: 97);an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 99); and/or an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 100), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 96-97 and 99-100, such as possessed by the anti-TIGIT antibodiesh6B2.L1H1, h6B2.L1H2, h6B2.L1H3, h6B2.L1H4, and h6B2.L1H5. In someinstances, the anti-TIGIT antibody includes an FR-L1 comprising theamino acid sequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 96); an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 98);an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 99); and/or an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 100), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 96 and 98-100, such as possessed by the anti-TIGIT antibodiesh6B2.L2H1 and h6B2.L2H5.

In some instances, the antibody further comprises at least one, two,three, or four of the following heavy chain variable region FRs: anFR-H1 comprising the amino acid sequence ofX₁VX₂LKESGPX₃X₄X₅X₆PX₇X₈TLX₉LTCX₁₀FSGFSLS (SEQ ID NO: 74), wherein X₁ isE or Q; X₂ is S or T; X₃ is A or G; X₄ is I or L; X₅ is L or V; X₆ is Kor Q; X₇ is S or T; X₈ is H or Q; X₉ is S or T; and X₁₀ is S or T; anFR-H2 comprising the amino acid sequence of WIRQPX₁X₂KX₃LEWLA (SEQ IDNO: 75), wherein X₁ is P or S; X₂ is E or G; and X₃ is A or G; an FR-H3comprising the amino acid sequence ofRLTX₁X₂KDX₃SX₄X₅QX₆X₇LX₈X₉TX₁₀X₁₁DX₁₂X₁₃DTATYYCAH (SEQ ID NO: 76),wherein X₁ is I or V; X₂ is S or T; X₃ is A or T; X₄ is K or N; X₅ is Dor N; X₆ is A or V; X₇ is F or V; X₈ is N or T; X₉ is M or V; X₁₀ is Nor S; X₁₁ is M or V; X₁₂ is P or T; and X₁₃ is T or V; and/or an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 77), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 74-77, such as possessed by the anti-TIGIT antibody rat6B2 andderivatives thereof (e.g., h6B2.L1H1, h6B2.L2H1, h6B2.L1H2, h6B2.L1H3,h6B2.L1H4, h6B2.L1H5, and h6B2.L2H5). The anti-TIGIT antibody mayfurther include, for example, at least one, two, three, or four of thefollowing heavy chain variable region FRs: an FR-H1 comprising the aminoacid sequence of QVSLKESGPGILQPSHTLSLTCSFSGFSLS (SEQ ID NO: 78); anFR-H2 comprising the amino acid sequence of WIRQPSEKGLEWLA (SEQ ID NO:79); an FR-H3 comprising the amino acid sequence ofRLTVSKDASNDQAFLNVTSVDTTDTATYYCAH (SEQ ID NO: 80); and/or an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 77), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 77-80, such as possessed by the anti-TIGIT antibody rat6B2. In someinstances, the anti-TIGIT antibody includes an FR-H1 comprising theamino acid sequence of EVTLKESGPALVKPTQTLTLTCTFSGFSLS (SEQ ID NO: 81);an FR-H2 comprising the amino acid sequence of WIRQPPGKALEWLA (SEQ IDNO: 82); an FR-H3 comprising the amino acid sequence ofRLTVTKDASKNQAVLTMTNMDPVDTATYYCAH (SEQ ID NO: 83); and/or an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 77), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 77 and 81-83, such as possessed by the anti-TIGIT antibodiesh6B2.L1H1 and h6B2.L2H1. In some instances, the anti-TIGIT antibodyincludes an FR-H1 comprising the amino acid sequence ofEVTLKESGPALVKPTQTLTLTCTFSGFSLS (SEQ ID NO: 81); an FR-H2 comprising theamino acid sequence of WIRQPPGKALEWLA (SEQ ID NO: 82); an FR-H3comprising the amino acid sequence of RLTITKDASKNQAVLTMTNMDPVDTATYYCAH(SEQ ID NO: 84); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 77), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 77, 81-82, and 84, such aspossessed by the anti-TIGIT antibody h6B2.L1H2. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVTLKESGPALVKPTQTLTLTCTFSGFSLS (SEQ ID NO: 81); an FR-H2 comprisingthe amino acid sequence of WIRQPPGKALEWLA (SEQ ID NO: 82); an FR-H3comprising the amino acid sequence of RLTVTKDTSKNQAVLTMTNMDPVDTATYYCAH(SEQ ID NO: 85); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 77), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 77, 81-82, and 85, such aspossessed by the anti-TIGIT antibody h6B2.L1H3. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVTLKESGPALVKPTQTLTLTCTFSGFSLS (SEQ ID NO: 81); an FR-H2 comprisingthe amino acid sequence of WIRQPPGKALEWLA (SEQ ID NO: 82); an FR-H3comprising the amino acid sequence of RLTVTKDASKNQVVLTMTNMDPVDTATYYCAH(SEQ ID NO: 86); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 77), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 77, 81-82, and 85, such aspossessed by the anti-TIGIT antibody h6B2.L1H4. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVTLKESGPALVKPTQTLTLTCTFSGFSLS (SEQ ID NO: 81); an FR-H2 comprisingthe amino acid sequence of WIRQPPGKALEWLA (SEQ ID NO: 82); an FR-H3comprising the amino acid sequence of RLTITKDTSKNQVVLTMTNMDPVDTATYYCAH(SEQ ID NO: 87); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 77), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 77, 81-82, and 87, such aspossessed by the anti-TIGIT antibodies h6B2.L1H5 and h6B2.L2H5.

In another example, the invention provides anti-TIGIT antibodies thatinclude at least one, two, three, four, five, or six HVRs selected from(a) an HVR-H1 comprising the amino acid sequence of TYGMGVS (SEQ ID NO:110); (b) an HVR-H2 comprising the amino acid sequence ofSIWWNGNTYYNPSLRS (SEQ ID NO: 111); (c) an HVR-H3 comprising the aminoacid sequence of TGGAVITWFAY (SEQ ID NO: 112); (d) an HVR-L1 comprisingthe amino acid sequence of KASQSVGKNIA (SEQ ID NO: 113); (e) an HVR-L2comprising the amino acid sequence of YASNRYT (SEQ ID NO: 114); and/or(f) an HVR-L3 comprising the amino acid sequence of QHIYNSPYP (SEQ IDNO: 115), or a combination of one or more of the above HVRs and one ormore variants thereof having at least about 90% sequence identity (e.g.,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any oneof SEQ ID NOs: 110-115. In some instances, any of the above anti-TIGITantibodies of the preceding example may include, for example, (a) anHVR-H1 comprising the amino acid sequence of TYGMGVS (SEQ ID NO: 110);(b) an HVR-H2 comprising the amino acid sequence of SIWWNGNTYYNPSLRS(SEQ ID NO: 111); (c) an HVR-H3 comprising the amino acid sequence ofTGGAVITWFAY (SEQ ID NO: 112); (d) an HVR-L1 comprising the amino acidsequence of KASQSVGKNIA (SEQ ID NO: 113); (e) an HVR-L2 comprising theamino acid sequence of YASNRYT (SEQ ID NO: 114); and (f) an HVR-L3comprising the amino acid sequence of QHIYNSPYP (SEQ ID NO: 115), suchas possessed by the anti-TIGIT antibody rat10A5 and derivatives thereof(e.g., h10A5.L1H1; h10A5.L2H1; h10A5.L3H1; h10A5.L4H1; h10A5.L1H2;h10A5.L1H3; h10A5.L1H4; and h10A5.L4H4).

In some instances, the antibody further comprises at least one, two,three, or four of the following light chain variable region frameworkregions (FRs): an FR-L1 comprising the amino acid sequence ofX₁IVMTQSPX₂X₃X₄SX₅SX₆GX₇RX₈TX₉X₁₀C (SEQ ID NO: 129), wherein X₁ is E orN; X₂ is A or K; X₃ is S or T; X₄ is L or M; X₅ is I or V; X₆ is I or P;X₇ is D or E; X₈ is A or V; X₉ is L or M; and X₁₀ is N or S; an FR-L2comprising the amino acid sequence of WYQQKX₁GQX₂PX₃LLIY (SEQ ID NO:130), wherein X₁ is P or T; X₂ is A or S; and X₃ is Q or R; an FR-L3comprising the amino acid sequence ofGX₁PX₂RFX₃GX₄GSGTX₅FTLTIX₆SX₇QX₈EDX₉AX₁₀X₁₁YC (SEQ ID NO: 131), whereinX₁ is I or V; X₂ is A or D; X₃ is S or T; X₄ is G or S; X₅ is D or E; X₆is N or S; X₇ is L or V; X₈ is A or S; X₉ is A or F; X₁₀ is F or V; andX₁₁ is F or Y; and/or an FR-L4 comprising the amino acid sequence ofFGX₁GTKX₂EIK (SEQ ID NO: 132), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 129-132, such as possessed bythe anti-TIGIT antibody rat10A5 and derivatives thereof (e.g.,h10A5.L1H1; h10A5.L2H1; h10A5.L3H1; h10A5.L4H1; h10A5.L1H2; h10A5.L1H3;h10A5.L1H4; and h10A5.L4H4). The anti-TIGIT antibody may furtherinclude, for example, at least one, two, three, or four of the followinglight chain variable region FRs: an FR-L1 comprising the amino acidsequence of NIVMTQSPKSMSISIGDRVTMNC (SEQ ID NO: 133); an FR-L2comprising the amino acid sequence of WYQQKTGQSPQLLIY (SEQ ID NO: 134);an FR-L3 comprising the amino acid sequence ofGVPDRFTGGGSGTDFTLTINSVQAEDAAFFYC (SEQ ID NO: 135); and/or an FR-L4comprising the amino acid sequence of FGTGTKLEIK (SEQ ID NO: 136), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 133-136, such as possessed by the anti-TIGIT antibody rat10A5. Insome instances, the anti-TIGIT antibody includes an FR-L1 comprising theamino acid sequence of EIVMTQSPATLSVSPGERATLSC (SEQ ID NO: 137); anFR-L2 comprising the amino acid sequence of WYQQKPGQSPRLLIY (SEQ ID NO:138); an FR-L3 comprising the amino acid sequence ofGVPARFSGSGSGTEFTLTISSLQSEDFAVYYC (SEQ ID NO: 140); and/or an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 142), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 137-138, 140, and 142, such as possessed by the anti-TIGITantibodies h10A5.L1H1, h10A5.L1H2, h10A5.L1H3, and h10A5.L1H4. In someinstances, the anti-TIGIT antibody includes an FR-L1 comprising theamino acid sequence of EIVMTQSPATLSVSPGERATLSC (SEQ ID NO: 137); anFR-L2 comprising the amino acid sequence of WYQQKPGQAPRLLIY (SEQ ID NO:139); an FR-L3 comprising the amino acid sequence ofGVPARFSGSGSGTEFTLTISSLQSEDFAVYYC (SEQ ID NO: 140); and/or an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 142), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 137, 139-140, and 142, such as possessed by the anti-TIGIT antibodyh10A5.L2H1. In some instances, the anti-TIGIT antibody includes an FR-L1comprising the amino acid sequence of EIVMTQSPATLSVSPGERATLSC (SEQ IDNO: 137); an FR-L2 comprising the amino acid sequence of WYQQKPGQSPRLLIY(SEQ ID NO: 138); an FR-L3 comprising the amino acid sequence ofGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC (SEQ ID NO: 141); and/or an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 142), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 137-138 and 141-142, such as possessed by the anti-TIGIT antibodiesh10A5.L3H1. In some instances, the anti-TIGIT antibody includes an FR-L1comprising the amino acid sequence of EIVMTQSPATLSVSPGERATLSC (SEQ IDNO: 137); an FR-L2 comprising the amino acid sequence of WYQQKPGQAPRLLIY(SEQ ID NO: 139); an FR-L3 comprising the amino acid sequence ofGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC (SEQ ID NO: 141); and/or an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 142), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 137, 139, and 141-142, such as possessed by the anti-TIGITantibodies h10A5.L4H1 and h10A5.L4H4.

In some instances, the antibody further comprises at least one, two,three, or four of the following heavy chain variable region FRs: FR-H1comprising the amino acid sequence ofX₁VX₂LKESGPX₃X₄X₅X₆PX₇X₈TLX₉LTCX₁₀FSGFSLT (SEQ ID NO: 116), wherein X₁is E or Q; X₂ is S or T; X₃ is A or G; X₄ is I or L; X₅ is L or V; X₆ isK or Q; X₇ is S or T; X₈ is H or Q; X₉ is S or T; and X₁₀ is S or T; anFR-H2 comprising the amino acid sequence of WIRQPX₁X₂KX₃LEWLA (SEQ IDNO: 117), wherein X₁ is P or S; X₂ is E or G; and Xs is A or G; an FR-H3comprising the amino acid sequence ofRLTX₁X₂KDTSX₃X₄QX₅X₆LX₇X₈TX₉X₁₀DX₁₁X₁₂DTATYYCAH (SEQ ID NO: 118),wherein X₁ is I or V; X₂ is S or T; X₃ is K or N; X₄ is D or N; X₅ is Aor V; X₆ is F or V; X₇ is N or T; X₈ is M or V; X₉ is N or S; X₁₀ is Mor V; X₁₁ is P or T; and X₁₂ is T or V; and/or an FR-H4 comprising theamino acid sequence of WGQGTLVTVSS (SEQ ID NO: 119), or a combination ofone or more of the above FRs and one or more variants thereof having atleast about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity) to any one of SEQ ID NOs: 116-119, suchas possessed by the anti-TIGIT antibody rat10A5 and derivatives thereof(e.g., h10A5.L1H1; h10A5.L2H1; h10A5.L3H1; h10A5.L4H1; h10A5.L1H2;h10A5.L1H3; h10A5.L1H4; and h10A5.L4H4). The anti-TIGIT antibody mayfurther include, for example, at least one, two, three, or four of thefollowing heavy chain variable region FRs: an FR-H1 comprising the aminoacid sequence of QVSLKESGPGILQPSHTLSLTCSFSGFSLT (SEQ ID NO: 120); anFR-H2 comprising the amino acid sequence of WIRQPSEKGLEWLA (SEQ ID NO:121); an FR-H3 comprising the amino acid sequence ofRLTVSKDTSNDQAFLNVTSVDTTDTATYYCAH (SEQ ID NO: 122); and/or an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 119), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 119-122, such as possessed by the anti-TIGIT antibody rat10A5. Insome instances, the anti-TIGIT antibody includes an FR-H1 comprising theamino acid sequence of EVTLKESGPALVKPTQTLTLTCTFSGFSLT (SEQ ID NO: 123);an FR-H2 comprising the amino acid sequence of WIRQPPGKALEWLA (SEQ IDNO: 124); an FR-H3 comprising the amino acid sequence ofRLTVTKDTSKNQAVLTMTNMDPVDTATYYCAH (SEQ ID NO: 125); and/or an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 119), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 119 and 123-125, such as possessed by the anti-TIGIT antibodiesh10A5.L1H1, h10A5.L2H1, h10A5.L3H1, and h10A5.L4H1. In some instances,the anti-TIGIT antibody includes an FR-H1 comprising the amino acidsequence of EVTLKESGPALVKPTQTLTLTCTFSGFSLT (SEQ ID NO: 123); an FR-H2comprising the amino acid sequence of WIRQPPGKALEWLA (SEQ ID NO: 124);an FR-H3 comprising the amino acid sequence ofRLTITKDTSKNQAVLTMTNMDPVDTATYYCAH (SEQ ID NO: 126); and/or an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 119), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 119, 123-124, and 126, such as possessed by the anti-TIGIT antibodyh10A5.L1H2. In some instances, the anti-TIGIT antibody includes an FR-H1comprising the amino acid sequence of EVTLKESGPALVKPTQTLTLTCTFSGFSLT(SEQ ID NO: 123); an FR-H2 comprising the amino acid sequence ofWIRQPPGKALEWLA (SEQ ID NO: 124); an FR-H3 comprising the amino acidsequence of RLTVTKDTSKNQVVLTMTNMDPVDTATYYCAH (SEQ ID NO: 127); and/or anFR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO:119), or a combination of one or more of the above FRs and one or morevariants thereof having at least about 90% sequence identity (e.g., 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one ofSEQ ID NOs: 119, 123-124, and 127, such as possessed by the anti-TIGITantibody h10A5.L1H3. In some instances, the anti-TIGIT antibody includesan FR-H1 comprising the amino acid sequence ofEVTLKESGPALVKPTQTLTLTCTFSGFSLT (SEQ ID NO: 123); an FR-H2 comprising theamino acid sequence of WIRQPPGKALEWLA (SEQ ID NO: 124); an FR-H3comprising the amino acid sequence of RLTITKDTSKNQVVLTMTNMDPVDTATYYCAH(SEQ ID NO: 128); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 119), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 119, 123-124, and 128, such aspossessed by the anti-TIGIT antibodies h10A5.L1H4 and h10A5.L4H4.

In another example, the invention provides anti-TIGIT antibodies thatinclude at least one, two, three, four, five, or six HVRs selected from(a) an HVR-H1 comprising the amino acid sequence of EYSIY (SEQ ID NO:153); (b) an HVR-H2 comprising the amino acid sequence ofRIDPKNGRTYYVDKFKN (SEQ ID NO: 154); (c) an HVR-H3 comprising the aminoacid sequence of IYGFYFDF (SEQ ID NO: 155); (d) an HVR-L1 comprising theamino acid sequence of KGSQNVNKYLV (SEQ ID NO: 156); (e) an HVR-L2comprising the amino acid sequence of NTDNLQS (SEQ ID NO: 157); and/or(f) an HVR-L3 comprising the amino acid sequence of YQYNNGFT (SEQ ID NO:158), or a combination of one or more of the above HVRs and one or morevariants thereof having at least about 90% sequence identity (e.g., 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one ofSEQ ID NOs: 153-158. In some instances, any of the above anti-TIGITantibodies of the preceding example may include, for example, (a) anHVR-H1 comprising the amino acid sequence of EYSIY (SEQ ID NO: 153); (b)an HVR-H2 comprising the amino acid sequence of RIDPKNGRTYYVDKFKN (SEQID NO: 154); (c) an HVR-H3 comprising the amino acid sequence ofIYGFYFDF (SEQ ID NO: 155); (d) an HVR-L1 comprising the amino acidsequence of KGSQNVNKYLV (SEQ ID NO: 156); (e) an HVR-L2 comprising theamino acid sequence of NTDNLQS (SEQ ID NO: 157); and (f) an HVR-L3comprising the amino acid sequence of YQYNNGFT (SEQ ID NO: 158) such aspossessed by the anti-TIGIT antibody rat7E7 and derivatives thereof(e.g., h7E7.L1H1, h7E7.L2H1, h7E7.L3H1, h7E7.L4H1, h7E7.L5H1, h7E7.L1H2,h7E7.L1H3, h7E7.L1H4, h7E7.L1H5, h7E7.L1H6, h7E7.L1H7, h7E7.L1H8,h7E7.L1H9, h7E7.L5H9, and 7E7.L5aH9a).

In some instances, the antibody further comprises at least one, two,three, or four of the following light chain variable region frameworkregions (FRs): an FR-L1 comprising the amino acid sequence ofX₁IX₂LTQSPSX₃LSASVGDRVTX₄X₅C (SEQ ID NO: 180), wherein X₁ is D or N; X₂is H or Q; X₃ is F or L; X₄ is I or L; and X₅ is S or T; an FR-L2comprising the amino acid sequence of WYQQKX₁GX₂APKLLIY (SEQ ID NO:181), wherein X₁ is L or P; and X₂ is E or K; an FR-L3 comprising theamino acid sequence of GX₁PSRFSGSGSGTX₂X₃TLTISSLQPEDX₄ATYX₅C (SEQ ID NO:182), wherein X₁ is I or V; X₂ is D or E; X₃ is F or Y; X₄ is A or F;and X₅ is F or Y; and/or an FR-L4 comprising the amino acid sequence ofFGX₁GTKX₂EIK (SEQ ID NO: 183), wherein X₁ is Q or S; and X₂ is L or V ora combination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 180-183, such as possessed by the anti-TIGIT antibody rat7E7 andderivatives thereof (e.g., h7E7.L1H1, h7E7.L2H1, h7E7.L3H1, h7E7.L4H1,h7E7.L5H1, h7E7.L1H2, h7E7.L1H3, h7E7.L1H4, h7E7.L1H5, h7E7.L1H6,h7E7.L1H7, h7E7.L1H8, h7E7.L1H9, h7E7.L5H9, and 7E7.L5aH9a). Theanti-TIGIT antibody may further include, for example, at least one, two,three, or four of the following light chain variable region FRs: anFR-L1 comprising the amino acid sequence of NIHLTQSPSLLSASVGDRVTLSC (SEQID NO: 184); an FR-L2 comprising the amino acid sequence ofWYQQKLGEAPKLLIY (SEQ ID NO: 185); an FR-L3 comprising the amino acidsequence of GIPSRFSGSGSGTDYTLTISSLQPEDAATYFC (SEQ ID NO: 186); and/or anFR-L4 comprising the amino acid sequence of FGSGTKLEIK (SEQ ID NO: 187),or a combination of one or more of the above FRs and one or morevariants thereof having at least about 90% sequence identity (e.g., 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one ofSEQ ID NOs: 184-187, such as possessed by the anti-TIGIT antibodyrat7E7. In some instances, the anti-TIGIT antibody includes an FR-L1comprising the amino acid sequence of DIQLTQSPSFLSASVGDRVTITC (SEQ IDNO: 188); an FR-L2 comprising the amino acid sequence of WYQQKPGKAPKLLIY(SEQ ID NO: 189); an FR-L3 comprising the amino acid sequence ofGIPSRFSGSGSGTEYTLTISSLQPEDFATYFC (SEQ ID NO: 190); and/or an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 196), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 188-190 and 196, such as possessed by the anti-TIGIT antibodiesh7E7.L1H1, h7E7.L1H2, h7E7.L1H3, h7E7.L1H4, h7E7.L1H5, h7E7.L1H6,h7E7.L1H7, h7E7.L1H8, and h7E7.L1H9. In some instances, the anti-TIGITantibody includes an FR-L1 comprising the amino acid sequence ofDIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 188); an FR-L2 comprising the aminoacid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 189); an FR-L3 comprisingthe amino acid sequence of GVPSRFSGSGSGTEYTLTISSLQPEDFATYFC (SEQ ID NO:191); and/or an FR-L4 comprising the amino acid sequence of FGQGTKVEIK(SEQ ID NO: 196), or a combination of one or more of the above FRs andone or more variants thereof having at least about 90% sequence identity(e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) toany one of SEQ ID NOs: 188-189, 191 and 196, such as possessed by theanti-TIGIT antibody h7E7.L2H1. In some instances, the anti-TIGITantibody includes an FR-L1 comprising the amino acid sequence ofDIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 188); an FR-L2 comprising the aminoacid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 189); an FR-L3 comprisingthe amino acid sequence of GIPSRFSGSGSGTEFTLTISSLQPEDFATYFC (SEQ ID NO:192); and/or an FR-L4 comprising the amino acid sequence of FGQGTKVEIK(SEQ ID NO: 196), or a combination of one or more of the above FRs andone or more variants thereof having at least about 90% sequence identity(e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) toany one of SEQ ID NOs: 188-189, 192 and 196, such as possessed by theanti-TIGIT antibodies h7E7.L3H1. In some instances, the anti-TIGITantibody includes an FR-L1 comprising the amino acid sequence ofDIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 188); an FR-L2 comprising the aminoacid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 189); an FR-L3 comprisingthe amino acid sequence of GIPSRFSGSGSGTEYTLTISSLQPEDFATYYC (SEQ ID NO:193); and/or an FR-L4 comprising the amino acid sequence of FGQGTKVEIK(SEQ ID NO: 196), or a combination of one or more of the above FRs andone or more variants thereof having at least about 90% sequence identity(e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) toany one of SEQ ID NOs: 188-189, 193 and 196, such as possessed by theanti-TIGIT antibodies h7E7.L4H1. In some instances, the anti-TIGITantibody includes an FR-L1 comprising the amino acid sequence ofDIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 188); an FR-L2 comprising the aminoacid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 189); an FR-L3 comprisingthe amino acid sequence of GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO:194); and/or an FR-L4 comprising the amino acid sequence of FGQGTKVEIK(SEQ ID NO: 196), or a combination of one or more of the above FRs andone or more variants thereof having at least about 90% sequence identity(e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) toany one of SEQ ID NOs: 188-189, 194 and 196, such as possessed by theanti-TIGIT antibodies h7E7.L5H1 and h7E7.L5H9. In some instances, theanti-TIGIT antibody includes an FR-L1 comprising the amino acid sequenceof DIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 188); an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 189); an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTEYTLTISSLQPEDFATYYC(SEQ ID NO: 195); and/or an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 196), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 188-189 and 195-196, such aspossessed by the anti-TIGIT antibodies 7E7.L5aH9a.

In some instances, the antibody further comprises at least one, two,three, or four of the following heavy chain variable region FRs: anFR-H1 comprising the amino acid sequence ofEVQLX₁QSGX₂EX₃X₄X₅PGASVKX₆SCKAX₇GYTFT (SEQ ID NO: 159), wherein X₁ is Qor V; X₂ is A or P; X₃ is L or V; X₄ is K or Q; X₅ is K or R; X₆ is L orV; and X₇ is S or T; an FR-H2 comprising the amino acid sequence ofWVX₁QX₂PX₃QX₄LEX₅X₆G (SEQ ID NO: 160), wherein X₁ is K or R; X₂ is A orR; X₃ is G or K; X₄ is R or S; X₅ is I or W; and X₆ is I or M; an FR-H3comprising the amino acid sequence ofRX₁TX₂TX₃DTSX₄X₅TAYMX₆LSSLX₇SEDTAX₈YX₉CX₁₀R (SEQ ID NO: 161), wherein X₁is A or V; X₂ is I or L; X₃ is A or R; X₄ is A or S; X₅ is N or S; X₆ isE or Q; X₇ is R or T; X₈ is T or V; X₉ is F or Y; and X₁₀ is A or T;and/or an FR-H4 comprising the amino acid sequence of WGQGX₁X₂VTX₃SS(SEQ ID NO: 162), wherein X₁ is T or V; X₂ is L or M; and X₃ is A or V,or a combination of one or more of the above FRs and one or morevariants thereof having at least about 90% sequence identity (e.g., 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one ofSEQ ID NOs: 159-162, such as possessed by the anti-TIGIT antibody rat7E7and derivatives thereof (e.g., h7E7.L1H1, h7E7.L2H1, h7E7.L3H1,h7E7.L4H1, h7E7.L5H1, h7E7.L1H2, h7E7.L1H3, h7E7.L1H4, h7E7.L1H5,h7E7.L1H6, h7E7.L1H7, h7E7.L1H8, h7E7.L1H9, h7E7.L5H9, and 7E7.L5aH9a).The anti-TIGIT antibody may further include, for example, at least one,two, three, or four of the following heavy chain variable region FRs: anFR-H1 comprising the amino acid sequence ofEVQLQQSGPELQRPGASVKLSCKATGYTFT (SEQ ID NO: 163); an FR-H2 comprising theamino acid sequence of WVKQRPKQSLEIIG (SEQ ID NO: 164); an FR-H3comprising the amino acid sequence of RATLTADTSSNTAYMQLSSLTSEDTATYFCTR(SEQ ID NO: 165); and/or an FR-H4 comprising the amino acid sequence ofWGQGVMVTASS (SEQ ID NO: 166), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 163-166, such as possessed bythe anti-TIGIT antibody rat7E7. In some instances, the anti-TIGITantibody includes an FR-H1 comprising the amino acid sequence ofEVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 167); an FR-H2 comprising theamino acid sequence of WVRQAPGQRLEIIG (SEQ ID NO: 168); an FR-H3comprising the amino acid sequence of RATLTADTSASTAYMELSSLRSEDTAVYFCTR(SEQ ID NO: 172); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 179), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 167-138, 172, and 179, such aspossessed by the anti-TIGIT antibodies h7E7.L1H1, h7E7.L2H1, h7E7.L3H1,h7E7.L4H1, and h7E7.L5H1. In some instances, the anti-TIGIT antibodyincludes an FR-H1 comprising the amino acid sequence ofEVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 167); an FR-H2 comprising theamino acid sequence of WVRQAPGQRLEWIG (SEQ ID NO: 169); an FR-H3comprising the amino acid sequence of RATLTADTSASTAYMELSSLRSEDTAVYFCTR(SEQ ID NO: 172); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 179), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 167, 169, 172, and 179, such aspossessed by the anti-TIGIT antibody h7E7.L1H2. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 167); an FR-H2 comprisingthe amino acid sequence of WVRQAPGQRLEIMG (SEQ ID NO: 170); an FR-H3comprising the amino acid sequence of RATLTADTSASTAYMELSSLRSEDTAVYFCTR(SEQ ID NO: 172); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 179), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 167, 170, 172, and 179, such aspossessed by the anti-TIGIT antibody h7E7.L1H3. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 167); an FR-H2 comprisingthe amino acid sequence of WVRQAPGQRLEIIG (SEQ ID NO: 168); an FR-H3comprising the amino acid sequence of RVTLTADTSASTAYMELSSLRSEDTAVYFCTR(SEQ ID NO: 173); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 179), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 167-168, 173, and 179, such aspossessed by the anti-TIGIT antibody h7E7.L1H4. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 167); an FR-H2 comprisingthe amino acid sequence of WVRQAPGQRLEIIG (SEQ ID NO: 168); an FR-H3comprising the amino acid sequence of RATITADTSASTAYMELSSLRSEDTAVYFCTR(SEQ ID NO: 174); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 179), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 167-168, 174, and 179, such aspossessed by the anti-TIGIT antibody h7E7.L1H5. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 167); an FR-H2 comprisingthe amino acid sequence of WVRQAPGQRLEIIG (SEQ ID NO: 168); an FR-H3comprising the amino acid sequence of RATLTRDTSASTAYMELSSLRSEDTAVYFCTR(SEQ ID NO: 175); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 179), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 167-168, 175, and 179, such aspossessed by the anti-TIGIT antibody h7E7.L1H6. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 167); an FR-H2 comprisingthe amino acid sequence of WVRQAPGQRLEIIG (SEQ ID NO: 168); an FR-H3comprising the amino acid sequence of RATLTADTSASTAYMELSSLRSEDTAVYYCTR(SEQ ID NO: 176); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 179), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 167-168, 176, and 179, such aspossessed by the anti-TIGIT antibody h7E7.L1H7. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 167); an FR-H2 comprisingthe amino acid sequence of WVRQAPGQRLEIIG (SEQ ID NO: 168); an FR-H3comprising the amino acid sequence of RATLTADTSASTAYMELSSLRSEDTAVYFCAR(SEQ ID NO: 177); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 179), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 167-168, 177, and 179, such aspossessed by the anti-TIGIT antibody h7E7.L1H8. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 167); an FR-H2 comprisingthe amino acid sequence of WVRQAPGQRLEWMG (SEQ ID NO: 171); an FR-H3comprising the amino acid sequence of RVTITRDTSASTAYMELSSLRSEDTAVYYCAR(SEQ ID NO: 178); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 179), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 167, 171, and 178-179, such aspossessed by the anti-TIGIT antibodies h7E7.L1H9 and h7E7.L5H9. In someinstances, the anti-TIGIT antibody includes an FR-H1 comprising theamino acid sequence of EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 167);an FR-H2 comprising the amino acid sequence of WVRQAPGQRLEIMG (SEQ IDNO: 170); an FR-H3 comprising the amino acid sequence ofRVTITRDTSASTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 178); and/or an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 179), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 167, 170, and 178-179, such as possessed by the anti-TIGIT antibody7E7.L5aH9a.

In another example, the invention provides anti-TIGIT antibodies thatinclude at least one, two, three, four, five, or six HVRs selected from(a) an HVR-H1 comprising the amino acid sequence of EHSIY (SEQ ID NO:215); (b) an HVR-H2 comprising the amino acid sequence ofRIDPKNGRTYFVDKFKN (SEQ ID NO: 216); (c) an HVR-H3 comprising the aminoacid sequence of IDGFYFDF (SEQ ID NO: 217); (d) an HVR-L1 comprising theamino acid sequence of KGSQNVNKYLV (SEQ ID NO: 218); (e) an HVR-L2comprising the amino acid sequence of STDNLQS (SEQ ID NO: 219); and/or(f) an HVR-L3 comprising the amino acid sequence of YQYNNGFT (SEQ ID NO:220), or a combination of one or more of the above HVRs and one or morevariants thereof having at least about 90% sequence identity (e.g., 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one ofSEQ ID NOs: 215-220. In some instances, any of the above anti-TIGITantibodies of the preceding example may include, for example, (a) anHVR-H1 comprising the amino acid sequence of EHSIY (SEQ ID NO: 215); (b)an HVR-H2 comprising the amino acid sequence of RIDPKNGRTYFVDKFKN (SEQID NO: 216); (c) an HVR-H3 comprising the amino acid sequence ofIDGFYFDF (SEQ ID NO: 217); (d) an HVR-L1 comprising the amino acidsequence of KGSQNVNKYLV (SEQ ID NO: 218); (e) an HVR-L2 comprising theamino acid sequence of STDNLQS (SEQ ID NO: 219); and (f) an HVR-L3comprising the amino acid sequence of YQYNNGFT (SEQ ID NO: 220), such aspossessed by the anti-TIGIT antibody rat1508 and derivatives thereof(e.g., h15C8.L1H1, h15C8.L2H1, h15C8.L3H1, h15C8.L4H1, h15C8.L5H1,h15C8.L1H2, h15C8.L1H3, h15C8.L1H4, h15C8.L1H5, h15C8.L1H6, h15C8.L1H7,h15C8.L1H8, h15C8.L1H9, h15C8.L5H9, and 5C8.L5aH9a).

In some instances, the antibody further comprises at least one, two,three, or four of the following light chain variable region frameworkregions (FRs): an FR-L1 comprising the amino acid sequence ofX₁IX₂LTQSPSX₃LSASVGDRVTX₄X₅C (SEQ ID NO: 243), wherein X₁ is D or N; X₂is H or Q; X₃ is F or L; X₄ is I or L; and X₅ is S or T; an FR-L2comprising the amino acid sequence of WYQQKX₁GX₂APKLLIY (SEQ ID NO:244), wherein X₁ is L or P and X₂ is E or K; an FR-L3 comprising theamino acid sequence of GX₁PSRFSGSGSGTX₂X₃TLTISSLQPEDX₄ATYX₅C (SEQ ID NO:245), wherein X₁ is I or V; X₂ is D or E; X₃ is F or Y; X₄ is A or F;and X₅ is F or Y; and/or an FR-L4 comprising the amino acid sequence ofFGX₁GTKX₂EIK (SEQ ID NO: 246), wherein X₁ is Q or S and X₂ is L or V, ora combination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 243-246, such as possessed by the anti-TIGIT antibody rat1508 andderivatives thereof (e.g., h15C8.L1H1, h15C8.L2H1, h15C8.L3H1,h15C8.L4H1, h15C8.L5H1, h15C8.L1H2, h15C8.L1H3, h15C8.L1H4, h15C8.L1H5,h15C8.L1H6, h15C8.L1H7, h15C8.L1H8, h15C8.L1H9, h15C8.L5H9, and5C8.L5aH9a). The anti-TIGIT antibody may further include, for example,at least one, two, three, or four of the following light chain variableregion FRs: an FR-L1 comprising the amino acid sequence ofNIHLTQSPSLLSASVGDRVTLSC (SEQ ID NO: 247); an FR-L2 comprising the aminoacid sequence of WYQQKLGEAPKLLIY (SEQ ID NO: 248); an FR-L3 comprisingthe amino acid sequence of GIPSRFSGSGSGTDYTLTISSLQPEDAATYFC (SEQ ID NO:249); and/or an FR-L4 comprising the amino acid sequence of FGSGTKLEIK(SEQ ID NO: 250), or a combination of one or more of the above FRs andone or more variants thereof having at least about 90% sequence identity(e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) toany one of SEQ ID NOs: 247-250, such as possessed by the anti-TIGITantibody rat1508. In some instances, the anti-TIGIT antibody includes anFR-L1 comprising the amino acid sequence of DIQLTQSPSFLSASVGDRVTITC (SEQID NO: 251); an FR-L2 comprising the amino acid sequence ofWYQQKPGKAPKLLIY (SEQ ID NO: 252); an FR-L3 comprising the amino acidsequence of GIPSRFSGSGSGTEYTLTISSLQPEDFATYFC (SEQ ID NO: 253); and/or anFR-L4 comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 259),or a combination of one or more of the above FRs and one or morevariants thereof having at least about 90% sequence identity (e.g., 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one ofSEQ ID NOs: 251-253 and 259, such as possessed by the anti-TIGITantibodies h15C8.L1H1, h15C8.L1H2, h15C8.L1H3, h15C8.L1H4, h15C8.L1H5,h15C8.L1H6, h15C8.L1H7, h15C8.L1H8, and h15C8.L1H9. In some instances,the anti-TIGIT antibody includes an FR-L1 comprising the amino acidsequence of DIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 251); an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 252);an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTEYTLTISSLQPEDFATYFC (SEQ ID NO: 254); and/or an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 259), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 251-252, 254, and 259, such as possessed by the anti-TIGIT antibodyh15C8.L2H1. In some instances, the anti-TIGIT antibody includes an FR-L1comprising the amino acid sequence of DIQLTQSPSFLSASVGDRVTITC (SEQ IDNO: 251); an FR-L2 comprising the amino acid sequence of WYQQKPGKAPKLLIY(SEQ ID NO: 252); an FR-L3 comprising the amino acid sequence ofGIPSRFSGSGSGTEFTLTISSLQPEDFATYFC (SEQ ID NO: 255); and/or an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 259), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 251-252, 255, and 259, such as possessed by the anti-TIGITantibodies h15C8.L3H1. In some instances, the anti-TIGIT antibodyincludes an FR-L1 comprising the amino acid sequence ofDIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 251); an FR-L2 comprising the aminoacid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 252); an FR-L3 comprisingthe amino acid sequence of GIPSRFSGSGSGTEYTLTISSLQPEDFATYYC (SEQ ID NO:256); and/or an FR-L4 comprising the amino acid sequence of FGQGTKVEIK(SEQ ID NO: 259), or a combination of one or more of the above FRs andone or more variants thereof having at least about 90% sequence identity(e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) toany one of SEQ ID NOs: 251-252, 256, and 259, such as possessed by theanti-TIGIT antibodies h15C8.L4H1. In some instances, the anti-TIGITantibody includes an FR-L1 comprising the amino acid sequence ofDIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 251); an FR-L2 comprising the aminoacid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 252); an FR-L3 comprisingthe amino acid sequence of GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO:257); and/or an FR-L4 comprising the amino acid sequence of FGQGTKVEIK(SEQ ID NO: 259), or a combination of one or more of the above FRs andone or more variants thereof having at least about 90% sequence identity(e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) toany one of SEQ ID NOs: 251-252, 257, and 259, such as possessed by theanti-TIGIT antibodies h15C8.L5H1 and h15C8.L5H9. In some instances, theanti-TIGIT antibody includes an FR-L1 comprising the amino acid sequenceof DIQLTQSPSFLSASVGDRVTITC (SEQ ID NO: 251); an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 252); an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTEYTLTISSLQPEDFATYYC(SEQ ID NO: 258); and/or an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 259), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 251-252, and 258-259, such aspossessed by the anti-TIGIT antibodies 5C8.L5aH9a. In some instances,the antibody further comprises at least one, two, three, or four of thefollowing heavy chain variable region FRs: FR-H1 comprising the aminoacid sequence of EVQLX₁QSGX₂EX₃X₄X₅PGASVKX₆SCKASGYTFT (SEQ ID NO: 221),wherein X₁ is Q or V; X₂ is A or P; X₃ is L or V; X₄ is K or Q; X₅ is Kor R; and Xs is L or V; an FR-H2 comprising the amino acid sequence ofWX₁X₂QX₃PX₄QX₅LEX₆X₇G (SEQ ID NO: 222), wherein X₁ is L or V; X₂ is K orR; X₃ is A or R; X₄ is G or K; X₅ is R or S; X₆ is I or W; and X₇ is Ior M; an FR-H3 comprising the amino acid sequence ofRX₁TX₂TX₃X₄TSX₅X₆TAYMX₇LSSLX₈SEDTAX₉YX₁₀CAR (SEQ ID NO: 223), wherein X₁is A or V; X₂ is I or L; X₃ is R or T; X₄ is D or N; X₅ is A or S; X₆ isN or S; X₇ is E or Q; X₈ is R or T; X₉ is I or V; and X₁₀ is F or Y;and/or an FR-H4 comprising the amino acid sequence of WGQGX₁X₂VTX₃SS(SEQ ID NO: 224), or a combination of one or more of the above FRs andone or more variants thereof having at least about 90% sequence identity(e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) toany one of SEQ ID NOs: 221-224, such as possessed by the anti-TIGITantibody rat15C8 and derivatives thereof (e.g., h15C8.L1H1, h15C8.L2H1,h15C8.L3H1, h15C8.L4H1, h15C8.L5H1, h15C8.L1H2, h15C8.L1H3, h15C8.L1H4,h15C8.L1H5, h15C8.L1H6, h15C8.L1H7, h15C8.L1H8, h15C8.L1H9, h15C8.L5H9,and 5C8.L5aH9a). The anti-TIGIT antibody may further include, forexample, at least one, two, three, or four of the following heavy chainvariable region FRs: an FR-H1 comprising the amino acid sequence ofEVQLQQSGPELQRPGASVKLSCKASGYTFT (SEQ ID NO: 225); an FR-H2 comprising theamino acid sequence of WLKQRPKQSLEIIG (SEQ ID NO: 226); an FR-H3comprising the amino acid sequence of RATLTTNTSSNTAYMQLSSLTSEDTAIYFCAR(SEQ ID NO: 227); and/or an FR-H4 comprising the amino acid sequence ofWGQGVMVTASS (SEQ ID NO: 228), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 225-228, such as possessed bythe anti-TIGIT antibody rat1508. In some instances, the anti-TIGITantibody includes an FR-H1 comprising the amino acid sequence ofEVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 229); an FR-H2 comprising theamino acid sequence of WLRQAPGQRLEIIG (SEQ ID NO: 230); an FR-H3comprising the amino acid sequence of RATLTTDTSASTAYMELSSLRSEDTAVYFCAR(SEQ ID NO: 235); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 242), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 229-230, 235, and 242, such aspossessed by the anti-TIGIT antibodies h15C8.L1H1, h15C8.L2H1,h15C8.L3H1, h15C8.L4H1, and h15C8.L5H1. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 229); an FR-H2 comprisingthe amino acid sequence of WVRQAPGQRLEIIG (SEQ ID NO: 231); an FR-H3comprising the amino acid sequence of RATLTTDTSASTAYMELSSLRSEDTAVYFCAR(SEQ ID NO: 235); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 242), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 229, 231, 235, and 242, such aspossessed by the anti-TIGIT antibody h15C8.L1H2. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 229); an FR-H2 comprisingthe amino acid sequence of WLRQAPGQRLEWIG (SEQ ID NO: 232); an FR-H3comprising the amino acid sequence of RATLTTDTSASTAYMELSSLRSEDTAVYFCAR(SEQ ID NO: 235); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 242), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 229, 232, 235, and 242, such aspossessed by the anti-TIGIT antibody h15C8.L1H3. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 229); an FR-H2 comprisingthe amino acid sequence of WLRQAPGQRLEIMG (SEQ ID NO: 233); an FR-H3comprising the amino acid sequence of RATLTTDTSASTAYMELSSLRSEDTAVYFCAR(SEQ ID NO: 235); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 242), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 229, 233, 235, and 242, such aspossessed by the anti-TIGIT antibody h15C8.L1H4. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 229); an FR-H2 comprisingthe amino acid sequence of WLRQAPGQRLEIIG (SEQ ID NO: 230); an FR-H3comprising the amino acid sequence of RVTLTTDTSASTAYMELSSLRSEDTAVYFCAR(SEQ ID NO: 236); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 242), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 229-230, 236, and 242, such aspossessed by the anti-TIGIT antibody h15C8.L1H5. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 229); an FR-H2 comprisingthe amino acid sequence of WLRQAPGQRLEIIG (SEQ ID NO: 230); an FR-H3comprising the amino acid sequence of RATITTDTSASTAYMELSSLRSEDTAVYFCAR(SEQ ID NO: 237); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 242), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 229-230, 237, and 242, such aspossessed by the anti-TIGIT antibody h15C8.L1H6. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 229); an FR-H2 comprisingthe amino acid sequence of WLRQAPGQRLEIIG (SEQ ID NO: 230); an FR-H3comprising the amino acid sequence of RATLTRDTSASTAYMELSSLRSEDTAVYFCAR(SEQ ID NO: 238); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 242), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 229-230, 238, and 242, such aspossessed by the anti-TIGIT antibody h15C8.L1H7. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 229); an FR-H2 comprisingthe amino acid sequence of WLRQAPGQRLEIIG (SEQ ID NO: 230); an FR-H3comprising the amino acid sequence of RATLTTDTSASTAYMELSSLRSEDTAVYYCAR(SEQ ID NO: 239); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 242), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 229-230, 239, and 242, such aspossessed by the anti-TIGIT antibody h15C8.L1H8. In some instances, theanti-TIGIT antibody includes an FR-H1 comprising the amino acid sequenceof EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 229); an FR-H2 comprisingthe amino acid sequence of WVRQAPGQRLEWMG (SEQ ID NO: 234); an FR-H3comprising the amino acid sequence of RVTITRDTSASTAYMELSSLRSEDTAVYYCAR(SEQ ID NO: 240); and/or an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 242), or a combination of one or more of theabove FRs and one or more variants thereof having at least about 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to any one of SEQ ID NOs: 229, 234, 240, and 242, such aspossessed by the anti-TIGIT antibodies h15C8.L1H9 and h15C8.L5H9. Insome instances, the anti-TIGIT antibody includes an FR-H1 comprising theamino acid sequence of EVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 229);an FR-H2 comprising the amino acid sequence of WLRQAPGQRLEIMG (SEQ IDNO: 233); an FR-H3 comprising the amino acid sequence ofRATITTDTSASTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 241); and/or an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 242), or acombination of one or more of the above FRs and one or more variantsthereof having at least about 90% sequence identity (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to any one of SEQ IDNOs: 229, 233, and 241-242, such as possessed by the anti-TIGIT antibody5C8.L5aH9a.

Anti-TIGIT antibodies are also provided that include a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 60, 62, 66, 101, 103, 104, 105, 106, 107, 145,146, 147, 148, 197, 199, 200, 201, 202, 203, 204, 205, 206, 207, 213,260, 262, 263, 264, 265, 266, 267, 268, 269, 270, or 276. Also providedare anti-TIGIT antibodies that include a light chain variable regionhaving at least 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95%(e.g., 96%, 97%, 98%, or 99%) sequence identity to, or the sequence of,SEQ ID NO: 61, 63, 64, 67, 102, 108, 109, 144, 149, 150, 151, 152, 198,208, 209, 210, 211, 212, 214, 261, 271, 272, 273, 274, 275, or 277.

In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 60, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:61. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 62, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:63. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 62, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:64. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 66, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:67. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 101, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:102. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 103, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:108. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 104, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:108. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 105, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:108. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 106, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:108. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 107, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:109. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 143, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:144. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 145, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:149. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 145, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:150. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 145, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:151. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 145, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:152. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 147, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:149. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 148, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:149. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 148, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:152. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 197, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:198. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 199, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:208. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 200, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:208. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 201, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:208. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 202, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:208. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 203, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:208. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 204, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:208. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 205, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:208. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 206, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:208. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 207, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:208. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 207, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:212. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 199, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:209. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 199, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:210. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 199, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:211. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 199, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:212. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 213, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:214. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 260, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:261. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 262, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:271. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 263, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:271. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 264, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:271. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 265, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:271. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 266, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:271. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 267, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:271. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 268, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:271. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 269, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:271. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 270, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:271. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 270, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:275. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 262, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:272. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 262, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:273. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 262, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:274. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 262, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:275. In some instances, the anti-TIGIT antibody includes a heavy chainvariable region having at least 90% (e.g., 91%, 92%, 93%, or 94%), or atleast 95% (e.g., 96%, 97%, 98%, or 99%) sequence identity to, or thesequence of, SEQ ID NO: 276, and a light chain variable region having atleast 90% (e.g., 91%, 92%, 93%, or 94%), or at least 95% (e.g., 96%,97%, 98%, or 99%) sequence identity to, or the sequence of, SEQ ID NO:277.

In another aspect, an anti-TIGIT antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above, wherein one or both ofthe variable domain sequences include post-translational modifications.

In some instances, any one of the anti-TIGIT antibodies described abovemay be capable of binding to rabbit TIGIT, in addition to human TIGIT.In some instances, any one of the anti-TIGIT antibodies described abovemay be capable of binding to both human TIGIT and cynomolgus monkey(cyno) TIGIT. In some instances, any one of the anti-TIGIT antibodiesdescribed above may be capable of binding to human TIGIT, cyno TIGIT,and rabbit TIGIT. In some instances, any one of the anti-TIGITantibodies described above (e.g., 4.1D3 or a derivative thereof) may becapable of binding to human TIGIT, cyno TIGIT, and rabbit TIGIT, but notmurine TIGIT.

In some instances, the anti-TIGIT antibody binds human TIGIT with a Kdof about 10 nM or lower and cyno TIGIT with a Kd of about 10 nM or lower(e.g., binds human TIGIT with a Kd of about 0.1 nM to about 1 nM andcyno TIGIT with a Kd of about 0.5 nM to about 1 nM, e.g., binds humanTIGIT with a Kd of about 0.1 nM or lower and cyno TIGIT with a Kd ofabout 0.5 nM or lower).

In some embodiments, the anti-TIGIT antibody is an antagonist antibody.The antagonist antibody may specifically bind TIGIT and inhibit or blockTIGIT interaction with poliovirus receptor (PVR) (e.g., the antagonistantibody inhibits intracellular signaling mediated by TIGIT binding toPVR). In some instances, the antagonist antibody inhibits or blocksbinding of human TIGIT to human PVR with an IC50 value of 10 nM or lower(e.g., 1 nM to about 10 nM). In some instances, the antagonist antibodyinhibits or blocks binding of cyno TIGIT to cyno PVR with an IC50 valueof 50 nM or lower (e.g., 1 nM to about 50 nM, e.g., 1 nM to about 5 nM).

In other embodiments, the anti-TIGIT antibody can be an agonistantibody. The agonist antibody can specifically bind TIGIT and stimulatethe interaction of PVR with CD226 or CD96. For example, the agonistantibody can specifically bind TIGIT and stimulate the interaction ofPVR with CD226 and CD96 (e.g., binds human TIGIT and stimulates theinteraction of human PVR with human CD226 and human CD96 and/or bindscyno TIGIT and stimulates the interaction of cyno PVR with cyno CD226and cyno CD96).

In a further aspect, the invention provides an isolated antibody thatcompetes for binding to TIGIT with any of the anti-TIGIT antibodiesdescribed above. In yet a further aspect, the invention provides anisolated antibody that binds to the same epitope as an anti-TIGITantibody described above.

An anti-TIGIT antibody according to any of the above embodiments is amonoclonal antibody, comprising a chimeric, humanized, or humanantibody. In one embodiment, an anti-TIGIT antibody is an antibodyfragment, for example, a Fv, Fab, Fab′, scFv, diabody, or F(ab′)2fragment. In another embodiment, the antibody is a full-length antibody,e.g., an intact IgG antibody (e.g., an intact IgG1 antibody) or otherantibody class or isotype as defined herein.

In a further aspect, an anti-TIGIT antibody according to any of theabove embodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below.

1. Antibody Affinity

In certain embodiments, an antibody (e.g., an anti-TIGIT antibody)provided herein has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸ M or less, e.g.,from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA). In one embodiment, an RIA is performed with the Fab versionof an antibody of interest and its antigen. For example, solutionbinding affinity of Fabs for antigen is measured by equilibrating Fabwith a minimal concentration of (¹²⁵I)-labeled antigen in the presenceof a titration series of unlabeled antigen, then capturing bound antigenwith an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol.Biol. 293:865-881(1999)). To establish conditions for the assay,MICROTITER® multi-well plates (Thermo Scientific) are coated overnightwith 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mMsodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovineserum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150μl/well of scintillant (MICROSCINT-20™; Packard) is added, and theplates are counted on a TOPCOUNT™ gamma counter (Packard) for tenminutes. Concentrations of each Fab that give less than or equal to 20%of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using a BIACORE® surfaceplasmon resonance assay. For example, an assay using a BIACORE®-2000 ora BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) is performed at 25° C.with immobilized antigen CM5 chips at ˜10 response units (RU). In oneembodiment, carboxymethylated dextran biosensor chips (CM5, BIACORE,Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN-20¹M) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μl/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). See, for example, Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 10⁶M⁻¹s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)2, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al. Nat. Med. 9:129-134 (2003); and Hollinger et al. Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al. Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing specificity determining region(SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J. Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In any one of the above aspects, the anti-TIGIT antibodies (e.g., 4.1D3or a variant thereof, e.g., 4.1D3.Q1E) provided herein can bemultispecific antibodies, for example, bispecific antibodies.Multispecific antibodies can be monoclonal antibodies that have bindingspecificities for at least two different sites. In certain embodiments,bispecific antibodies may bind to two different epitopes on TIGIT. Incertain embodiments, one of the binding specificities is for TIGIT andthe other is for any other antigen (e.g., a second biological molecule,e.g., a cell surface antigen, e.g., a tumor antigen). Accordingly, abispecific anti-TIGIT antibody may have binding specificities for TIGITand a second biological molecule, such as a PD-1, PD-L1, PD-L2, OX40,PD-1, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, or CD96. Bispecificantibodies can also be prepared as full-length antibodies or antibodyfragments.

In other embodiments, bispecific antibodies may bind to two differentepitopes of TIGIT, OX40, PD-1, PD-L1, PD-L2. In certain embodiments, oneof the binding specificities is for TIGIT and the other is for any otherantigen (e.g., a second biological molecule, such as OX40). In otherembodiments, the bispecific antibody may have binding specificity forTIGIT and PD-L1; TIGIT and PD-L2; TIGIT and PD-1; TIGIT and CTLA-4;TIGIT and LAG3; TIGIT and TIM3; TIGIT and BTLA; TIGIT and VISTA; TIGITand B7H4; or TIGIT and CD96, wherein the bispecific antibody ispreferably an antagonist antibody for TIGIT and an antagonist antibodyfor its second target. In other embodiments, the bispecific antibody mayhave binding specificity for TIGIT and CD226; TIGIT and CD28; TIGIT andCD27; TIGIT and CD137; TIGIT and HVEM; TIGIT and GITR; TIGIT and MICA;TIGIT and ICOS; TIGIT and NKG2D; or TIGIT and 2B4, wherein thebispecific antibody is preferably an antagonist antibody for TIGIT andfor its second target. In other embodiments, the bispecific antibody mayhave binding specificity for TIGIT that is not antagonistic in nature(i.e., the bispecific antibody does not have act as a TIGIT antagonist).

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). “Knob-in-hole” engineering of multispecificantibodies may be utilized to generate a first arm containing a knob anda second arm containing the hole into which the knob of the first armmay bind. The knob of the multispecific antibodies of the invention maybe an anti-TIGIT arm in one embodiment. Alternatively, the knob of themultispecific antibodies of the invention may be an anti-TIGIT arm inone embodiment. Multispecific antibodies may also be engineered usingimmunoglobulin crossover (also known as Fab domain exchange or CrossMabformat) technology (see e.g., WO2009/080253; Schaefer et al., Proc.Natl. Acad. Sci. USA, 108:11187-11192 (2011)). Multispecific antibodiesmay also be made by engineering electrostatic steering effects formaking antibody Fc-heterodimeric molecules (WO 2009/089004A1);cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat.No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); usingleucine zippers to produce bispecific antibodies (see, e.g., Kostelny etal., J. Immunol., 148(5):1547-1553 (1992)); using “diabody” technologyfor making bispecific antibody fragments (see, e.g., Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chainFv (sFv) dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994));and preparing trispecific antibodies as described, e.g., in Tutt et al.J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibodies, or antibody fragments thereof, may also include a “DualActing FAb” or “DAF” comprising an antigen binding site that binds toTIGIT as well as another, different antigen (e.g., a second biologicalmolecule) (see, e.g., US 2008/0069820).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the anti-TIGITantibodies of the invention are contemplated. As described in detailherein, anti-TIGIT antibodies may be optimized based on desiredstructural and functional properties. For example, it may be desirableto improve the binding affinity and/or other biological properties ofthe antibody. Amino acid sequence variants of an antibody may beprepared by introducing appropriate modifications into the nucleotidesequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of residues within the amino acid sequences ofthe antibody. Any combination of deletion, insertion, and substitutioncan be made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, for example,antigen-binding.

I. Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, for example, retained/improved antigen binding,decreased immunogenicity, or improved ADCC or CDC.

TABLE 1 Exemplary and Preferred Amino Acid Substitutions OriginalExemplary Preferred Residue Substitutions Substitutions Ala (A) Val;Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; ArgGln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I)Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val;Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile LeuPhe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr ThrThr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser PheVal (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or residues that contact antigen,with the resulting variant VH or VL being tested for binding affinity.Affinity maturation by constructing and reselecting from secondarylibraries has been described, e.g., in Hoogenboom et al. in Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,N.J., (2001).) In some embodiments of affinity maturation, diversity isintroduced into the variable genes chosen for maturation by any of avariety of methods (e.g., error-prone PCR, chain shuffling, oroligonucleotide-directed mutagenesis). A secondary library is thencreated. The library is then screened to identify any antibody variantswith the desired affinity. Another method to introduce diversityinvolves HVR-directed approaches, in which several HVR residues (e.g.,4-6 residues at a time) are randomized. HVR residues involved in antigenbinding may be specifically identified, e.g., using alanine scanningmutagenesis or modeling. CDR-H3 and CDR-L3 in particular are oftentargeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

II. Glycosylation Variants

In certain embodiments, anti-TIGIT antibodies of the invention (e.g.,4.1D3 or a variant thereof, e.g., 4.1D3.Q1E) can be altered to increaseor decrease the extent to which the antibody is glycosylated. Additionor deletion of glycosylation sites to anti-TIGIT antibody of theinvention may be conveniently accomplished by altering the amino acidsequence such that one or more glycosylation sites is created orremoved.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, anti-TIGIT antibody variants are provided having acarbohydrate structure that lacks fucose attached (directly orindirectly) to an Fc region. For example, the amount of fucose in suchantibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from20% to 40%. The amount of fucose is determined by calculating theaverage amount of fucose within the sugar chain at Asn297, relative tothe sum of all glycostructures attached to Asn 297 (e.g. complex, hybridand high mannose structures) as measured by MALDI-TOF mass spectrometry,as described in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (EUnumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Anti-TIGIT antibodies variants are further provided with bisectedoligosaccharides, for example, in which a biantennary oligosaccharideattached to the Fc region of the antibody is bisected by GlcNAc. Suchantibody variants may have reduced fucosylation and/or improved ADCCfunction. Examples of such antibody variants are described, e.g., in WO2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana etal.); and US 2005/0123546 (Umana et al.). Antibody variants with atleast one galactose residue in the oligosaccharide attached to the Fcregion are also provided. Such antibody variants may have improved CDCfunction. Such antibody variants are described, e.g., in WO 1997/30087(Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

III. Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an anti-TIGIT antibody of the invention(e.g., 4.1D3 or a variant thereof, e.g., 4.1D3.Q1E), thereby generatingan Fc region variant (see e.g., US 2012/0251531). The Fc region variantmay comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3or IgG4 Fc region) comprising an amino acid modification (e.g., asubstitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates an anti-TIGITantibody variant that possesses some but not all effector functions,which make it a desirable candidate for applications in which the halflife of the antibody in vivo is important yet certain effector functions(such as complement and ADCC) are unnecessary or deleterious. In vitroand/or in vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. See, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al. J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al. Blood. 101:1045-1052 (2003); and Cragg, M. S. and M. J. GlennieBlood. 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al. Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. Nos. 6,737,056 and 8,219,149). Such Fcmutants include Fc mutants with substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine(U.S. Pat. Nos. 7,332,581 and 8,219,149).

In certain embodiments, the proline at position 329 of a wild-type humanFc region in the antibody is substituted with glycine or arginine or anamino acid residue large enough to destroy the proline sandwich withinthe Fc/Fc.gamma. receptor interface that is formed between the proline329 of the Fc and tryptophan residues Trp 87 and Trp 110 of FcgRIII(Sondermann et al.: Nature 406, 267-273 (20 Jul. 2000)). In certainembodiments, the antibody comprises at least one further amino acidsubstitution. In one embodiment, the further amino acid substitution isS228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S, and still inanother embodiment the at least one further amino acid substitution isL234A and L235A of the human IgG1 Fc region or S228P and L235E of thehuman IgG4 Fc region (see e.g., US 2012/0251531), and still in anotherembodiment the at least one further amino acid substitution is L234A andL235A and P329G of the human IgG1 Fc region.

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

In some aspects the anti-TIGIT antibody (e.g., 4.1D3 or a variantthereof, e.g., 4.1D3.Q1E) comprises an Fc region comprising an N297Gmutation.

In some embodiments, the anti-TIGIT antibody (e.g., 4.1D3 or a variantthereof, e.g., 4.1D3.Q1E) comprises one or more heavy chain constantdomains, wherein the one or more heavy chain constant domains areselected from a first CH1 (CH1₁) domain, a first CH2 (CH2₁) domain, afirst CH3 (CH3₁) domain, a second CH1 (CH1₂) domain, second CH2 (CH2₂)domain, and a second CH3 (CH3₂) domain. In some instances, at least oneof the one or more heavy chain constant domains is paired with anotherheavy chain constant domain. In some instances, the CH3₁ and CH3₂domains each comprise a protuberance or cavity, and wherein theprotuberance or cavity in the CH3₁ domain is positionable in the cavityor protuberance, respectively, in the CH3₂ domain. In some instances,the CH3₁ and CH3₂ domains meet at an interface between said protuberanceand cavity. In some instances, the CH2₁ and CH2₂ domains each comprise aprotuberance or cavity, and wherein the protuberance or cavity in theCH2₁ domain is positionable in the cavity or protuberance, respectively,in the CH2₂ domain. In other instances, the CH2₁ and CH2₂ domains meetat an interface between said protuberance and cavity. In some instances,the anti-TIGIT antibody is an IgG1 antibody.

IV. Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, for example, inU.S. Pat. No. 7,521,541.

V. Antibody Derivatives

In certain embodiments, an anti-TIGIT antibody of the invention (e.g.,4.1D3 or a variant thereof, e.g., 4.1D3.Q1E) provided herein may befurther modified to contain additional nonproteinaceous moieties thatare known in the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

IV. Recombinant Methods and Compositions

Anti-TIGIT antibodies of the invention (e.g., 4.1D3 or a variantthereof, e.g., 4.1D3.Q1E) may be produced using recombinant methods andcompositions, for example, as described in U.S. Pat. No. 4,816,567,which is incorporated herein by reference in its entirety. In oneembodiment, isolated nucleic acid encoding an anti-TIGIT antibodydescribed herein is provided. Such nucleic acid may encode an amino acidsequence comprising the VL and/or an amino acid sequence comprising theVH of the antibody (e.g., the light and/or heavy chains of theantibody). In a further embodiment, one or more vectors (e.g.,expression vectors) comprising such nucleic acid are provided. In afurther embodiment, a host cell comprising such nucleic acid isprovided. In one such embodiment, a host cell comprises (e.g., has beentransformed with): (1) a vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and an aminoacid sequence comprising the VH of the antibody, or (2) a first vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and a second vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VH of the antibody.In one embodiment, the host cell is eukaryotic, e.g. a Chinese HamsterOvary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In oneembodiment, a method of making an anti-TIGIT antibody is provided,wherein the method comprises culturing a host cell comprising a nucleicacid encoding the antibody, as provided above, under conditions suitablefor expression of the antibody, and optionally recovering the antibodyfrom the host cell (or host cell culture medium).

For recombinant production of an anti-TIGIT antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.,2003), pp. 245-254, describing expression of antibody fragments in E.coli.) After expression, the antibody may be isolated from the bacterialcell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

V. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-TIGITantibody of the invention (e.g., 4.1D3 or a variant thereof, e.g.,4.1D3.Q1E) conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an anti-TIGITantibody as described herein (e.g., 4.1D3 or a variant thereof, e.g.,4.1D3.Q1E) conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an anti-TIGITantibody as described herein (e.g., 4.1D3 or a variant thereof, e.g.,4.1D3.Q1E) conjugated to a radioactive atom to form a radioconjugate. Avariety of radioactive isotopes are available for the production ofradioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸,Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu. When theradioconjugate is used for detection, it may comprise a radioactive atomfor scintigraphic studies, for example tc99m or I123, or a spin labelfor nuclear magnetic resonance (NMR) imaging (also known as magneticresonance imaging, mri), such as iodine-123 again, iodine-131,indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

VI. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-TIGIT antibodies of theinvention (e.g., 4.1D3 or a variant thereof, e.g., 4.1D3.Q1E) is usefulfor detecting the presence of TIGIT in a biological sample. The term“detecting” as used herein encompasses quantitative or qualitativedetection. In certain embodiments, a biological sample comprises a cellor tissue.

In one embodiment, an anti-TIGIT antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of TIGIT in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-TIGIT antibody as described herein under conditionspermissive for binding of the anti-TIGIT antibody to TIGIT, anddetecting whether a complex is formed between the anti-TIGIT antibodyand TIGIT. Such method may be an in vitro or in vivo method.

In certain embodiments, labeled anti-TIGIT antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

VII. Pharmaceutical Compositions

Pharmaceutical compositions of an anti-TIGIT antibody of the invention(e.g., 4.1D3 or a variant thereof, e.g., 4.1D3.Q1E) are prepared bymixing such an antibody having the desired degree of purity with one ormore optional pharmaceutically acceptable carriers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized compositions or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

In some instances, a pharmaceutical composition including an anti-TIGITantibody provided herein may further include a PD-1 axis bindingantagonist, such as a PD-1 binding antagonist, a PD-L1 bindingantagonist, and a PD-L2 binding antagonist.

In some instances, a pharmaceutical composition including an anti-TIGITantibody provided herein may further include an OX40 binding agonist,such as an OX40 agonist antibody, an OX40L agonist fragment, an OX40oligomeric receptor, and an OX40 immunoadhesin.

In some instances, a pharmaceutical composition including an anti-TIGITantibody provided herein may further include an additional therapeuticagent, such as a chemotherapeutic agent or an agent that decreases orinhibits one or more additional immune co-inhibitory receptors (e.g.,PD-1, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, and/or CD96).

In other embodiments, a pharmaceutical composition including ananti-TIGIT antibody may further include (a) a PD-1 axis bindingantagonist and an OX40 binding agonist; (b) a PD-1 axis bindingantagonist and an additional therapeutic agent (e.g., a chemotherapeuticagent); (c) a PD-1 axis binding antagonist and an agent that decreasesor inhibits one or more additional immune co-inhibitory receptors (e.g.,PD-1, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, and/or CD96); (d) an OX40binding agonist and an additional therapeutic agent (e.g., achemotherapeutic agent); (e) an OX40 binding agonist and an agent thatdecreases or inhibits one or more additional immune co-inhibitoryreceptors (e.g., PD-1, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, and/orCD96); (f) an additional therapeutic agent (e.g., a chemotherapeuticagent) and an agent that decreases or inhibits one or more additionalimmune co-inhibitory receptors (e.g., PD-1, CTLA-4, LAG3, TIM3, BTLA,VISTA, B7H4, and/or CD96); or (g) a PD-1 axis binding antagonist, anOX40 binding agonist, and an agent that decreases or inhibits one ormore additional immune co-inhibitory receptors (e.g., PD-1, CTLA-4,LAG3, TIM3, BTLA, VISTA, B7H4, and/or CD96). Optionally, thepharmaceutical composition will include one or more pharmaceuticallyacceptable carriers, excipients, or diluent.

Exemplary lyophilized antibody compositions are described in U.S. Pat.No. 6,267,958. Aqueous antibody compositions include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter compositionsincluding a histidine-acetate buffer.

The composition herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, it may be desirable to further provide an additionaltherapeutic agent (e.g., a chemotherapeutic agent, a cytotoxic agent, agrowth inhibitory agent, and/or an anti-hormonal agent, such as thoserecited herein above). Such active ingredients are suitably present incombination in amounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, for example, films, or microcapsules.

The compositions to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

VIII. Therapeutic Methods

Any of the anti-TIGIT antibodies of the invention (e.g., 4.1D3 or avariant thereof, e.g., 4.1D3.Q1E) may be used in therapeutic methods.

In one aspect, an anti-TIGIT antibody for use as a medicament isprovided. In further aspects, an anti-TIGIT antibody described herein(e.g., 4.1D3 or a variant thereof, e.g., 4.1D3.Q1E) for use in treatingor delaying progression of an immune-related disease in a subject isprovided. In certain embodiments, the invention provides an anti-TIGITantibody for use in a method of treating a subject having animmune-related disease that is associated with a T cell dysfunctionaldisorder. In other embodiments, the immune-related disease is a viralinfection. In certain embodiments, the viral infection is a chronicviral infection. In some embodiments, T cell dysfunctional disorder ischaracterized by decreased responsiveness to antigenic stimulation. Insome embodiments, the T cell dysfunctional disorder is characterized byT cell anergy or decreased ability to secrete cytokines, proliferate orexecute cytolytic activity. In some embodiments, the T celldysfunctional disorder is characterized by T cell exhaustion. In someembodiments, the T cells are CD4+ and CD8+ T cells. In some embodiments,the T cell dysfunctional disorder includes unresolved acute infection,chronic infection and tumor immunity.

In another aspect, an anti-TIGIT antibody described herein (e.g., 4.1D3or a variant thereof, e.g., 4.1D3.Q1E) for treating or delayingprogression of a cancer in a subject is provided. In certainembodiments, the cancer is selected from the group consisting of anon-small cell lung cancer, a small cell lung cancer, a renal cellcancer, a colorectal cancer, an ovarian cancer, a breast cancer, apancreatic cancer, a gastric carcinoma, a bladder cancer, an esophagealcancer, a mesothelioma, a melanoma, a head and neck cancer, a thyroidcancer, a sarcoma, a prostate cancer, a glioblastoma, a cervical cancer,a thymic carcinoma, a leukemia, a lymphomas, a myeloma (e.g., multiplemyeloma (MM)), mycoses fungoides, a merkel cell cancer, and ahematologic malignancy. Thus, a variety of cancers may be treated, ortheir progression may be delayed. In some embodiments, the individualmay have breast cancer (e.g., triple-negative breast cancer). In otherembodiments, the individual may have pancreatic cancer (e.g., pancreaticductal adenocarcinoma (PDAC)). In some embodiments, the individual hasnon-small cell lung cancer. The non-small cell lung cancer may be atearly stage or at late stage. In some embodiments, the individual hassmall cell lung cancer. The small cell lung cancer may be at early stageor at late stage. In some embodiments, the individual has renal cellcancer. The renal cell cancer may be at early stage or at late stage. Insome embodiments, the individual has colorectal cancer. The colorectalcancer may be at early stage or late stage. In some embodiments, theindividual has ovarian cancer. The ovarian cancer may be at early stageor at late stage. In some embodiments, the individual has breast cancer.The breast cancer may be at early stage or at late stage. In someembodiments, the individual has pancreatic cancer. The pancreatic cancermay be at early stage or at late stage. In some embodiments, theindividual has gastric carcinoma. The gastric carcinoma may be at earlystage or at late stage. In some embodiments, the individual has bladdercancer. The bladder cancer may be at early stage or at late stage. Insome embodiments, the individual has esophageal cancer. The esophagealcancer may be at early stage or at late stage. In some embodiments, theindividual has mesothelioma. The mesothelioma may be at early stage orat late stage. In some embodiments, the individual has melanoma. Themelanoma may be at early stage or at late stage. In some embodiments,the individual has head and neck cancer. The head and neck cancer may beat early stage or at late stage. In some embodiments, the individual hasthyroid cancer. The thyroid cancer may be at early stage or at latestage. In some embodiments, the individual has sarcoma. The sarcoma maybe at early stage or late stage. In some embodiments, the individual hasprostate cancer. The prostate cancer may be at early stage or at latestage. In some embodiments, the individual has glioblastoma. Theglioblastoma may be at early stage or at late stage. In someembodiments, the individual has cervical cancer. The cervical cancer maybe at early stage or at late stage. In some embodiments, the individualhas thymic carcinoma. The thymic carcinoma may be at early stage or atlate stage. In some embodiments, the individual has leukemia. Theleukemia may be at early stage or at late stage. In some embodiments,the individual has lymphomas. The lymphoma may be at early stage or atlate stage. In some embodiments, the individual has a myeloma (e.g.,MM). The myeloma (e.g., MM) may be at early stage or at late stage. Insome embodiments, the individual has mycoses fungoides. The mycosesfungoides may be at early stage or at late stage. In some embodiments,the individual has merkel cell cancer. The merkel cell cancer may be atearly stage or at late stage. In some embodiments, the individual hashematologic malignancies. The hematological malignancies may be earlystage or late stage.

In another aspect, an anti-TIGIT antibody described herein (e.g., 4.1D3or a variant thereof, e.g., 4.1D3.Q1E) for use in increasing, enhancing,or stimulating an immune response or function in a subject in needthereof is provided. In some embodiments, the immune response orfunction is increased, enhanced, and/or stimulated by activatingeffector cells (e.g., T cells, e.g., CD8+ and/or CD4+ T cells),expanding (increasing) an effector cell population, and/or killingtarget cells (e.g., target tumor cells) in the subject. In someembodiments, the CD4 and/or CD8 T cells in the individual have increasedor enhanced priming, activation, proliferation, cytokine release and/orcytolytic activity relative to prior to the administration of thecombination. In some embodiments, the number of CD4 and/or CD8 T cellsis elevated relative to prior to administration of the combination. Insome embodiments, the number of activated CD4 and/or CD8 T cells iselevated relative to prior to administration of the combination. In someembodiments, the activated CD4 and/or CD8 T cells is characterized byγ-IFN⁺ producing CD4 and/or CD8 T cells and/or enhanced cytolyticactivity relative to prior to the administration of the combination. Insome embodiments, the CD4 and/or CD8 T cells exhibit increased releaseof cytokines selected from the group consisting of IFN-γ, TNF-α andinterleukins. In some embodiments of the methods of this invention, theCD4 and/or CD8 T cell is an effector memory T cell. In some embodiments,the CD4 and/or CD8 effector memory T cell is characterized by γ-IFN⁺producing CD4 and/or CD8 T cells and/or enhanced cytolytic activity. Insome embodiments, the CD4 and/or CD8 effector memory T cell ischaracterized by having the expression of CD44^(high) CD62L^(low). Insome embodiments, the cancer has elevated levels of T cell infiltration.

In any of the above aspects, the anti-TIGIT antibody may be for use incombination with a different TIGIT antagonist, a PD-1 axis bindingantagonist, a OX40 binding agonist, an agent that decreases or inhibitsone or more additional immune co-inhibitory receptors, and/or anadditional therapeutic agent, such as a chemotherapeutic agent, asdescribed in detail herein.

In another aspect, the invention provides for the use of an anti-TIGITantibody described herein (e.g., 4.1D3 or a variant thereof, e.g.,4.1D3.Q1E) in the manufacture or preparation of a medicament. In oneembodiment, the medicament is for treating or delaying progression of acancer. In a further embodiment, the medicament is for treating ordelaying progression of a non-small cell lung cancer, a small cell lungcancer, a renal cell cancer, a colorectal cancer, an ovarian cancer, abreast cancer, a pancreatic cancer, a gastric carcinoma, a bladdercancer, an esophageal cancer, a mesothelioma, a melanoma, a head andneck cancer, a thyroid cancer, a sarcoma, a prostate cancer, aglioblastoma, a cervical cancer, a thymic carcinoma, a leukemia, alymphoma, a myeloma, mycoses fungoides, a merkel cell cancer, or ahematologic malignancy. Thus, a variety of cancers may be treated, ortheir progression may be delayed. In some embodiments, the individualmay have breast cancer (e.g., triple-negative breast cancer). In otherembodiments, the individual may have pancreatic cancer (e.g., pancreaticductal adenocarcinoma (PDAC)). In some embodiments, the individual hasnon-small cell lung cancer. The non-small cell lung cancer may be atearly stage or at late stage. In some embodiments, the individual hassmall cell lung cancer. The small cell lung cancer may be at early stageor at late stage. In some embodiments, the individual has renal cellcancer. The renal cell cancer may be at early stage or at late stage. Insome embodiments, the individual has colorectal cancer. The colorectalcancer may be at early stage or late stage. In some embodiments, theindividual has ovarian cancer. The ovarian cancer may be at early stageor at late stage. In some embodiments, the individual has breast cancer.The breast cancer may be at early stage or at late stage. In someembodiments, the individual has pancreatic cancer. The pancreatic cancermay be at early stage or at late stage. In some embodiments, theindividual has gastric carcinoma. The gastric carcinoma may be at earlystage or at late stage. In some embodiments, the individual has bladdercancer. The bladder cancer may be at early stage or at late stage. Insome embodiments, the individual has esophageal cancer. The esophagealcancer may be at early stage or at late stage. In some embodiments, theindividual has mesothelioma. The mesothelioma may be at early stage orat late stage. In some embodiments, the individual has melanoma. Themelanoma may be at early stage or at late stage. In some embodiments,the individual has head and neck cancer. The head and neck cancer may beat early stage or at late stage. In some embodiments, the individual hasthyroid cancer. The thyroid cancer may be at early stage or at latestage. In some embodiments, the individual has sarcoma. The sarcoma maybe at early stage or late stage. In some embodiments, the individual hasprostate cancer. The prostate cancer may be at early stage or at latestage. In some embodiments, the individual has glioblastoma. Theglioblastoma may be at early stage or at late stage. In someembodiments, the individual has cervical cancer. The cervical cancer maybe at early stage or at late stage. In some embodiments, the individualhas thymic carcinoma. The thymic carcinoma may be at early stage or atlate stage. In some embodiments, the individual has leukemia. Theleukemia may be at early stage or at late stage. In some embodiments,the individual has lymphomas. The lymphoma may be at early stage or atlate stage. In some embodiments, the individual has a myeloma (e.g.,MM). The myeloma (e.g., MM) may be at early stage or at late stage. Insome embodiments, the individual has mycoses fungoides. The mycosesfungoides may be at early stage or at late stage. In some embodiments,the individual has merkel cell cancer. The merkel cell cancer may be atearly stage or at late stage. In some embodiments, the individual hashematologic malignancies. The hematological malignancies may be earlystage or late stage.

In another aspect, the invention provides for the use of an anti-TIGITantibody described herein (e.g., 4.1D3 or a variant thereof, e.g.,4.1D3.Q1E) in the manufacture of a medicament for treating or delayingprogression of an immune-related disease. In some embodiments, theimmune-related disease is associated with T cell dysfunctional disorder.In some embodiments, the immune-related disease is a viral infection. Incertain embodiments, the viral infection is a chronic viral infection.In some embodiments, T cell dysfunctional disorder is characterized bydecreased responsiveness to antigenic stimulation. In some embodiments,the T cell dysfunctional disorder is characterized by T cell anergy ordecreased ability to secrete cytokines, proliferate or execute cytolyticactivity. In some embodiments, the T cell dysfunctional disorder ischaracterized by T cell exhaustion. In some embodiments, the T cells areCD4+ and CD8+ T cells. In some embodiments, the T cell dysfunctionaldisorder includes unresolved acute infection, chronic infection andtumor immunity.

In another aspect, an anti-TIGIT antibody described herein (e.g., 4.1D3or a variant thereof, e.g., 4.1D3.Q1E) in the manufacture of amedicament for use in increasing, enhancing, or stimulating an immuneresponse or function in a subject in need thereof is provided. In someembodiments, the immune response or function is increased, enhanced,and/or stimulated by activating effector cells (e.g., T cells, e.g.,CD8+ and/or CD4+ T cells), expanding (increasing) an effector cellpopulation, and/or killing target cells (e.g., target tumor cells) inthe subject. In some embodiments, the CD4 and/or CD8 T cells in theindividual have increased or enhanced priming, activation,proliferation, cytokine release and/or cytolytic activity relative toprior to the administration of the combination. In some embodiments, thenumber of CD4 and/or CD8 T cells is elevated relative to prior toadministration of the combination. In some embodiments, the number ofactivated CD4 and/or CD8 T cells is elevated relative to prior toadministration of the combination. In some embodiments, the activatedCD4 and/or CD8 T cells is characterized by γ-IFN⁺ producing CD4 and/orCD8 T cells and/or enhanced cytolytic activity relative to prior to theadministration of the combination. In some embodiments, the CD4 and/orCD8 T cells exhibit increased release of cytokines selected from thegroup consisting of IFN-γ, TNF-α and interleukins. In some embodimentsof the methods of this invention, the CD4 and/or CD8 T cell is aneffector memory T cell. In some embodiments, the CD4 and/or CD8 effectormemory T cell is characterized by γ-IFN⁺ producing CD4 and/or CD8 Tcells and/or enhanced cytolytic activity. In some embodiments, the CD4and/or CD8 effector memory T cell is characterized by having theexpression of CD44^(high) CD62L^(low). In some embodiments, the cancerhas elevated levels of T cell infiltration.

In any of the above aspects, the medicament may be formulated for use incombination with a different TIGIT antagonist, a PD-1 axis bindingantagonist, an OX40 binding agonist, an agent that decreases or inhibitsone or more additional immune co-inhibitory receptors, and/or anadditional therapeutic agent, such as a chemotherapeutic agent, asdescribed in detail herein.

In another aspect, the invention provides a method for treating ordelaying progression of a cancer in a subject, the method comprisingadministering to the subject an effective amount of an anti-TIGITantibody described herein (e.g., 4.1D3 or a variant thereof, e.g.,4.1D3.Q1E), thereby treating or delaying the progression of the cancerin the subject. In certain embodiments, the cancer is selected from thegroup consisting of a non-small cell lung cancer, a small cell lungcancer, a renal cell cancer, a colorectal cancer, an ovarian cancer, abreast cancer, a pancreatic cancer, a gastric carcinoma, a bladdercancer, an esophageal cancer, a mesothelioma, a melanoma, a head andneck cancer, a thyroid cancer, a sarcoma, a prostate cancer, aglioblastoma, a cervical cancer, a thymic carcinoma, a leukemia, alymphoma, a myeloma, mycoses fungoides, a merkel cell cancer, and ahematologic malignancy. Thus, a variety of cancers may be treated, ortheir progression may be delayed. In some embodiments, the individualmay have breast cancer (e.g., triple-negative breast cancer). In otherembodiments, the individual may have pancreatic cancer (e.g., pancreaticductal adenocarcinoma (PDAC)). In some embodiments, the individual hasnon-small cell lung cancer. The non-small cell lung cancer may be atearly stage or at late stage. In some embodiments, the individual hassmall cell lung cancer. The small cell lung cancer may be at early stageor at late stage. In some embodiments, the individual has renal cellcancer. The renal cell cancer may be at early stage or at late stage. Insome embodiments, the individual has colorectal cancer. The colorectalcancer may be at early stage or late stage. In some embodiments, theindividual has ovarian cancer. The ovarian cancer may be at early stageor at late stage. In some embodiments, the individual has breast cancer.The breast cancer may be at early stage or at late stage. In someembodiments, the individual has pancreatic cancer. The pancreatic cancermay be at early stage or at late stage. In some embodiments, theindividual has gastric carcinoma. The gastric carcinoma may be at earlystage or at late stage. In some embodiments, the individual has bladdercancer. The bladder cancer may be at early stage or at late stage. Insome embodiments, the individual has esophageal cancer. The esophagealcancer may be at early stage or at late stage. In some embodiments, theindividual has mesothelioma. The mesothelioma may be at early stage orat late stage. In some embodiments, the individual has melanoma. Themelanoma may be at early stage or at late stage. In some embodiments,the individual has head and neck cancer. The head and neck cancer may beat early stage or at late stage. In some embodiments, the individual hasthyroid cancer. The thyroid cancer may be at early stage or at latestage. In some embodiments, the individual has sarcoma. The sarcoma maybe at early stage or late stage. In some embodiments, the individual hasprostate cancer. The prostate cancer may be at early stage or at latestage. In some embodiments, the individual has glioblastoma. Theglioblastoma may be at early stage or at late stage. In someembodiments, the individual has cervical cancer. The cervical cancer maybe at early stage or at late stage. In some embodiments, the individualhas thymic carcinoma. The thymic carcinoma may be at early stage or atlate stage. In some embodiments, the individual has leukemia. Theleukemia may be at early stage or at late stage. In some embodiments,the individual has lymphomas. The lymphoma may be at early stage or atlate stage. In some embodiments, the individual has a myeloma (e.g.,MM). The myeloma (e.g., MM) may be at early stage or at late stage. Insome embodiments, the individual has mycoses fungoides. The mycosesfungoides may be at early stage or at late stage. In some embodiments,the individual has merkel cell cancer. The merkel cell cancer may be atearly stage or at late stage. In some embodiments, the individual hashematologic malignancies. The hematological malignancies may be earlystage or late stage.

In another aspect, the invention provides a method for treating ordelaying progression of an immune-related disease in a subject, themethod comprising administering to the subject an effective amount of ananti-TIGIT antibody described herein (e.g., 4.1D3 or a variant thereof,e.g., 4.1D3.Q1E), thereby treating or delaying the progression of theimmune-related disease in the subject. In some embodiments, theimmune-related disease is associated with T cell dysfunctional disorder.In some embodiments, the immune-related disease is a viral infection. Incertain embodiments, the viral infection is a chronic viral infection.In some embodiments, T cell dysfunctional disorder is characterized bydecreased responsiveness to antigenic stimulation. In some embodiments,the T cell dysfunctional disorder is characterized by T cell anergy ordecreased ability to secrete cytokines, proliferate or execute cytolyticactivity. In some embodiments, the T cell dysfunctional disorder ischaracterized by T cell exhaustion. In some embodiments, the T cells areCD4+ and CD8+ T cells. In some embodiments, the T cell dysfunctionaldisorder includes unresolved acute infection, chronic infection andtumor immunity.

In another aspect, the invention provides a method of increasing,enhancing, or stimulating an immune response or function in a subject,the comprising administering to the subject an effective amount of ananti-TIGIT antibody described herein (e.g., 4.1D3 or a variant thereof,e.g., 4.1D3.Q1E), thereby increasing, enhancing, or stimulating animmune response or function in the subject. In some embodiments, theimmune response or function is increased, enhanced, and/or stimulated byactivating effector cells (e.g., T cells, e.g., CD8+ and/or CD4+ Tcells), expanding (increasing) an effector cell population, and/orkilling target cells (e.g., target tumor cells) in the subject. In someembodiments of the methods of this invention, the CD4 and/or CD8 T cellsin the individual have increased or enhanced priming, activation,proliferation, cytokine release and/or cytolytic activity relative toprior to the administration of the combination. In some embodiments ofthe methods of this invention, the number of CD4 and/or CD8 T cells iselevated relative to prior to administration of the combination. In someembodiments of the methods of this invention, the number of activatedCD4 and/or CD8 T cells is elevated relative to prior to administrationof the combination. In some embodiments of the methods of thisinvention, the activated CD4 and/or CD8 T cells is characterized byγ-IFN⁺ producing CD4 and/or CD8 T cells and/or enhanced cytolyticactivity relative to prior to the administration of the combination. Insome embodiments of the methods of this invention, the CD4 and/or CD8 Tcells exhibit increased release of cytokines selected from the groupconsisting of IFN-γ, TNF-α and interleukins. In some embodiments of themethods of this invention, the CD4 and/or CD8 T cell is an effectormemory T cell. In some embodiments of the methods of this invention, theCD4 and/or CD8 effector memory T cell is characterized by γ-IFN⁺producing CD4 and/or CD8 T cells and/or enhanced cytolytic activity. Insome embodiments of the methods of this invention, the CD4 and/or CD8effector memory T cell is characterized by having the expression ofCD44^(high) CD62L^(low). In some embodiments of the methods of thisinvention, the cancer has elevated levels of T cell infiltration.

In any of the above aspects, the anti-TIGIT antibody may be foradministration in combination with a different TIGIT antagonist, a PD-1axis binding antagonist, an OX40 binding agonist, an agent thatdecreases or inhibits one or more additional immune co-inhibitoryreceptors, and/or an additional therapeutic agent, such as achemotherapeutic agent, as described in detail herein.

In some embodiments, the different (second) TIGIT antagonist can be anantagonist of TIGIT expression and/or activity, such as a small moleculeinhibitor, an inhibitory antibody or antigen-binding fragment thereof,an aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.In some embodiments, the TIGIT antagonist is a different anti-TIGITantibody or antigen-binding fragment thereof (e.g., one that binds to adifferent epitope on TIGIT that is either non-overlapping or onlypartially overlapping with the epitope recognized by the anti-TIGITantibody of the invention that is used). In some embodiments, the TIGITantagonist is an inhibitory nucleic acid selected from an antisensepolynucleotide, an interfering RNA, a catalytic RNA, and an RNA-DNAchimera.

In some embodiments, TIGIT antagonist can be an agent that modulates theCD226 expression and/or activity. For example, an agent that modulatesthe CD226 expression and/or activity that is capable of increasingand/or stimulating CD226 expression and/or activity; increasing and/orstimulating the interaction of CD226 with PVR, PVRL2, and/or PVRL3; andincreasing and/or stimulating the intracellular signaling mediated byCD226 binding to PVR, PVRL2, and/or PVRL3. As used herein, an agent thatis capable of increasing and/or stimulating CD226 expression and/oractivity includes, without limitation, agents that increase and/orstimulate CD226 expression and/or activity. As used herein, an agentthat is capable of increasing and/or stimulating the interaction ofCD226 with PVR, PVRL2, and/or PVRL3 includes, without limitation, agentsthat increase and/or stimulate the interaction of CD226 with PVR, PVRL2,and/or PVRL3. As used herein, an agent that is capable of increasingand/or stimulating the intracellular signaling mediated by CD226 bindingto PVR, PVRL2, and/or PVRL3 includes, without limitation, agents thatincrease and/or stimulate the intracellular signaling mediated by CD226binding to PVR, PVRL2, and/or PVRL3.

In some embodiments, the agent that modulates the CD226 expressionand/or activity is selected from an agent that inhibits and/or blocksthe interaction of CD226 with TIGIT, an antagonist of PVR expressionand/or activity, an agent that inhibits and/or blocks the interaction ofTIGIT with PVR, an agent that inhibits and/or blocks the interaction ofTIGIT with PVRL2, an agent that inhibits and/or blocks the interactionof TIGIT with PVRL3, an agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVR, an agent thatinhibits and/or blocks the intracellular signaling mediated by TIGITbinding to PVRL2, an agent that inhibits and/or blocks the intracellularsignaling mediated by TIGIT binding to PVRL3, and combinations thereof.

In some embodiments, the agent that inhibits and/or blocks theinteraction of CD226 with TIGIT is a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide. In someembodiments, the agent that inhibits and/or blocks the interaction ofCD226 with TIGIT is an anti-TIGIT antibody or antigen-binding fragmentthereof (e.g., one that binds to a different epitope on TIGIT that iseither non-overlapping or only partially overlapping with the epitoperecognized by the anti-TIGIT antibody of the invention that is used). Insome embodiments, the agent that inhibits and/or blocks the interactionof CD226 with TIGIT is an inhibitory nucleic acid selected from anantisense polynucleotide, an interfering RNA, a catalytic RNA, and anRNA-DNA chimera.

In some embodiments, the antagonist of PVR expression and/or activity isa small molecule inhibitor, an inhibitory antibody or antigen-bindingfragment thereof, an aptamer, an inhibitory nucleic acid, and aninhibitory polypeptide. In some embodiments, the antagonist of PVRexpression and/or activity is selected from a small molecule inhibitor,an inhibitory antibody or antigen-binding fragment thereof, an aptamer,an inhibitory nucleic acid, and an inhibitory polypeptide.

In some embodiments, the agent that inhibits and/or blocks theinteraction of TIGIT with PVR is a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide. In someembodiments, the agent that inhibits and/or blocks the interaction ofTIGIT with PVR is selected from a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide. In someembodiments, the agent that inhibits and/or blocks the interaction ofTIGIT with PVRL2 is selected from a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide. In someembodiments, the agent that inhibits and/or blocks the interaction ofTIGIT with PVRL3 is selected from a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide.

In some embodiments, the agent that inhibits and/or blocks theintracellular signaling mediated by TIGIT binding to PVR is a smallmolecule inhibitor, an inhibitory antibody or antigen-binding fragmentthereof, an aptamer, an inhibitory nucleic acid, and an inhibitorypolypeptide. In some embodiments, the agent that inhibits and/or blocksthe intracellular signaling mediated by TIGIT binding to PVR is selectedfrom a small molecule inhibitor, an inhibitory antibody orantigen-binding fragment thereof, an aptamer, an inhibitory nucleicacid, and an inhibitory polypeptide. In some embodiments, the agent thatinhibits and/or blocks the intracellular signaling mediated by TIGITbinding to PVRL2 is selected from a small molecule inhibitor, aninhibitory antibody or antigen-binding fragment thereof, an aptamer, aninhibitory nucleic acid, and an inhibitory polypeptide. In someembodiments, the agent that inhibits and/or blocks the intracellularsignaling mediated by TIGIT binding to PVRL3 is selected from a smallmolecule inhibitor, an inhibitory antibody or antigen-binding fragmentthereof, an aptamer, an inhibitory nucleic acid, and an inhibitorypolypeptide.

Other TIGIT antagonists that can be used in combination with theanti-TIGIT antibodies of the invention include the anti-TIGIT antibodiesand compositions containing such antibodies described in WO 2009/126688,which is incorporated by reference herein in its entirety.

In some instances, the methods provided herein include administration ofan effective amount of a PD-1 axis binding antagonist, prior to,subsequent to, or concurrently with an anti-TIGIT antibody of theinvention (and, optionally, one or more additional agents, such as asecond, different TIGIT antagonist, an OX40 binding agonist, achemotherapeutic agent, etc.). The PD-1 axis binding antagonist can beselected from the group consisting of a PD-1 binding antagonist, a PD-L1binding antagonist, and a PD-L2 binding antagonist.

In some embodiments of the above aspect, the PD-1 axis bindingantagonist is a PD-1 binding antagonist. In some embodiments, the PD-1binding antagonist inhibits the binding of PD-1 to its ligand bindingpartners. In some embodiments, the PD-1 binding antagonist inhibits thebinding of PD-1 to PD-L1. In some embodiments, the PD-1 bindingantagonist inhibits the binding of PD-1 to PD-L2. In some embodiments,the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1and PD-L2. In some embodiments, the PD-1 binding antagonist is anantibody. In some embodiments, the PD-1 binding antagonist is selectedfrom the group consisting of MDX 1106 (nivolumab), MK-3475(pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001,REGN2810, and BGB-108.

In other embodiments of the above aspect, the PD-1 axis bindingantagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1binding antagonist inhibits the binding of PD-L1 to PD-1. In someembodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits thebinding of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1binding antagonist is an antibody. In some embodiments, the antibody isselected from the group consisting of: MPDL3280A (atezolizumab),YW243.55.S70, MDX-1105, MED14736 (durvalumab), and MSB0010718C(avelumab).

In other embodiments of the above aspect, the PD-1 axis bindingantagonist is a PD-L2 binding antagonist. In some embodiments, the PD-L2binding antagonist is an antibody. In some embodiments, the PD-L2binding antagonist is an immunoadhesin.

As a general proposition, the therapeutically effective amount of a PD-1axis binding antagonist (e.g., an anti-PD-L1 antibody) may beadministered to a human will be in the range of about 0.01 to about 50mg/kg of patient body weight whether by one or more administrations. Insome embodiments, for example, the antagonist (e.g., anti-PD-L1antibody) is administered in a dose of about 0.01 to about 45 mg/kg,about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01to about 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg administereddaily, for example. In some embodiments, the antagonist (e.g.,anti-PD-L1 antibody) is administered at 15 mg/kg. However, other dosageregimens may be useful. In one embodiment, a PD-1 axis bindingantagonist (e.g., anti-PD-L1 antibody) is administered to a human at adose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, orabout 1500 mg. In some embodiments, a PD-1 axis binding antagonist(e.g., anti-PD-L1 antibody) is administered at a dose of about 800 mg toabout 850 mg every two weeks. In some embodiments, a PD-1 axis bindingantagonist (e.g., anti-PD-L1 antibody) is administered at a dose ofabout 840 mg every two weeks. The dose may be administered as a singledose or as multiple doses (e.g., 2 or 3 doses), such as infusions. Thedose of the antibody administered in a combination treatment may bereduced as compared to a single treatment. In some embodiments, forexample, the method for treating or delaying progression of locallyadvanced or metastatic breast cancer in an individual comprises a dosingregimen comprising treatment cycles, wherein the individual isadministered, on days 1 and 15 of each cycle, a human PD-1 axis bindingantagonist (e.g., anti-PD-L1 antibody) at a dose of about 840 mg,wherein each cycle is 28 days (i.e., each cycle is repeated every 28days). The progress of this therapy is easily monitored by conventionaltechniques.

In some embodiments, the OX40 binding agonist includes, for example, anOX40 agonist antibody (e.g., an anti-human OX40 agonist antibody), anOX40L agonist fragment, an OX40 oligomeric receptor, and an OX40immunoadhesin.

In some embodiments, the OX40 agonist antibody depletes cells thatexpress human OX40 (e.g., CD4+ effector T cells, CD8+ T cells, and/orTreg cells), for example, by ADCC and/or phagocytosis. In someembodiments, the OX40 agonist antibody binds human OX40 with an affinityof less than or equal to about 1 nM (e.g., less than or equal to about0.5 nM, e.g., less than or equal to about 0.45 nM, e.g., less than orequal to about 0.4 nM, e.g., less than or equal to about 0.3 nM). Insome embodiments, the binding affinity of the OX40 agonist antibody isdetermined using radioimmunoassay.

In some embodiments, the OX40 agonist antibody binds human OX40 andcynomolgus OX40. In further embodiments, binding to human OX40 andcynomolgus OX40 is determined using a FACS assay. In some embodiments,binding to human OX40 has an EC50 of less than or equal to about 1 μg/ml(e.g., less than or equal to about 0.7 μg/ml, e.g., less than or equalto about 0.5 μg/ml, e.g., less than or equal to about 0.4 μg/ml, e.g.,less than or equal to about 0.3 μg/ml, e.g., less than or equal to about0.2 μg/ml, e.g., less than or equal to about 0.1 μg/ml). In someembodiments, binding to cynomolgus OX40 has an EC50 of less than orequal to 3 μg/ml (e.g., less than or equal to about 2 μg/ml, e.g., lessthan or equal to about 1.7 μg/ml, e.g., less than or equal to about 1.5μg/ml, e.g., less than or equal to about 1.4 μg/ml, e.g., less than orequal to about 1.3 μg/ml, e.g., less than or equal to about 1.2 μg/ml,e.g., less than or equal to about 1.1 μg/ml, e.g., less than or equal toabout 1.0 μg/ml).

In some embodiments, the OX40 agonist antibody increases CD4+ effector Tcell proliferation and/or increases cytokine production by the CD4+effector T cell as compared to proliferation and/or cytokine productionprior to treatment with the OX40 agonist antibody. In some embodiments,the cytokine is IFN-γ.

In some embodiments, the OX40 agonist antibody increases memory T cellproliferation and/or increasing cytokine production by the memory cell.In some embodiments, the cytokine is IFN-γ.

In some embodiments, the OX40 agonist antibody inhibits Treg suppressionof effector T cell function. In some embodiments, effector T cellfunction is effector T cell proliferation and/or cytokine production. Insome embodiments, the effector T cell is a CD4+ effector T cell.

In some embodiments, the OX40 agonist antibody increases OX40 signaltransduction in a target cell that expresses OX40. In some embodiments,OX40 signal transduction is detected by monitoring NFkB downstreamsignaling.

In some embodiments, the OX40 agonist antibody is stable after treatmentat 40° C. for one to four weeks, e.g., one week, two weeks, three weeks,or four weeks. In some embodiments, the OX40 agonist antibody is stableafter treatment at 40° C. for two weeks.

In some embodiments, the OX40 agonist antibody comprises a variant IgG1Fc polypeptide comprising a mutation that eliminates binding to humaneffector cells has diminished activity relative to the OX40 agonistantibody comprising a native sequence IgG1 Fc portion. In someembodiments, the OX40 agonist antibody comprises a variant Fc portioncomprising a DANA mutation.

In some embodiments, antibody cross-linking is required for anti-humanOX40 antagonist antibody function.

In some embodiments, the OX40 agonist antibody comprises (a) a VH domaincomprising (i) a HVR-H1 comprising the amino acid sequence of SEQ ID NO:278, 279, or 280, (ii) a HVR-H2 comprising the amino acid sequence ofSEQ ID NO: 281, 282, 283, 284, 285, or 286, and (iii) a HVR-H3comprising an amino acid sequence selected from SEQ ID NO: 287, 288, or289; and (b) a VL domain comprising (i) a HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 290, (ii) a HVR-L2 comprising the amino acidsequence of SEQ ID NO: 291, and (iii) a HVR-L3 comprising the amino acidsequence of SEQ ID NO: 292, 293, 294, 295, 296, 297, 298, or 299. Forexample, in some embodiments, the OX40 agonist antibody comprises (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 278; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 281; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 287; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 290; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 291; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO: 292. In someembodiments, the OX40 agonist antibody comprises (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 278; (b) HVR-H2 comprising theamino acid sequence of SEQ ID NO: 281; (c) HVR-H3 comprising the aminoacid sequence of SEQ ID NO: 287; (d) HVR-L1 comprising the amino acidsequence of SEQ ID NO: 290; (e) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 291; and (f) HVR-L3 comprising an amino acidsequence selected from SEQ ID NO: 297. In some embodiments, the OX40agonist antibody comprises (a) HVR-H1 comprising the amino acid sequenceof SEQ ID NO: 278; (b) HVR-H2 comprising the amino acid sequence of SEQID NO: 281; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:287; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 290;(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 291; and (f)HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 298.

In other embodiments, the OX40 agonist antibody comprises a VH sequencehaving at least 90% sequence identity to the amino acid sequence of anyone of SEQ ID NOs: 300-325. For example, the OX40 agonist antibodycomprises a VH sequence having at least 90% sequence identity to theamino acid sequence of SEQ ID NO: 300 (e.g., wherein a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO: 300). Thus, in some embodiments, the OX40 agonist antibody comprisesa VH comprising one, two, or three HVRs selected from: (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 278, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 281, and (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 287.

In other embodiments, the OX40 agonist antibody comprises a VL sequencehaving at least 90% sequence identity to the amino acid sequence of anyone of SEQ ID NOs: 326-351. For example, the OX40 agonist antibodycomprises a VL having at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 326 (e.g., wherein a total of 1 to 10 amino acidshave been substituted, inserted, and/or deleted in SEQ ID NO: 326).Thus, in some embodiments, the OX40 agonist antibody comprises a VLcomprising one, two, or three HVRs selected from (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 290; (b) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 291; and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 292.

In some embodiments, the OX40 agonist antibody comprises (a) a VHsequence of SEQ ID NO: 300; (b) a VL sequence of SEQ ID NO: 326; or (c)a VH sequence as in (a) and a VL sequence as in (b).

In some embodiments, the OX40 agonist antibody comprises (a) a VHsequence of SEQ ID NO: 319; (b) a VL sequence of SEQ ID NO: 345; or (c)a VH sequence as in (a) and a VL sequence as in (b).

In some embodiments, the OX40 agonist antibody comprises (a) a VHsequence of SEQ ID NO: 320; (b) a VL sequence of SEQ ID NO: 346; or (c)a VH sequence as in (a) and a VL sequence as in (b).

Also specifically contemplated are OX40 agonist antibodies, such as anantibody sharing the same or substantially the same HVR sequences and/orVH and VL sequences of the anti-OX40 antibodies disclosed in U.S. Pat.No. 7,550,140 and International Pub. Nos. WO 2014/148895 and WO2013/038191, which are incorporated herein by reference in theirentirety.

In some embodiments, the OX40 agonist antibody is L106 BD (PharmingenProduct #340420). In some embodiments, the antibody comprises at leastone, two, three, four, five, or six hypervariable region (HVR) sequencesof antibody L106 (BD Pharmingen Product #340420). In some embodiments,the antibody comprises a heavy chain variable region sequence and/or alight chain variable region sequence of antibody L106 (BD PharmingenProduct #340420).

In some embodiments the OX40 agonist antibody is ACT35 (Santa CruzBiotechnology, Catalog #20073). In some embodiments, the antibodycomprises at least one, two, three, four, five, or six hypervariableregion (HVR) sequences of antibody ACT35 (Santa Cruz Biotechnology,Catalog #20073). In some embodiments, the antibody comprises a heavychain variable region sequence and/or a light chain variable regionsequence of antibody ACT35 (Santa Cruz Biotechnology, Catalog #20073).

In some embodiments, the OX40 agonist antibody is MED16469. In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody MED16469.In some embodiments, the antibody comprises a heavy chain variableregion sequence and/or a light chain variable region sequence ofantibody MED16469.

In some embodiments, the OX40 agonist antibody is MED10562. In someembodiments, the antibody comprises at least one, two, three, four,five, or six hypervariable region (HVR) sequences of antibody MED10562.In some embodiments, the antibody comprises a heavy chain variableregion sequence and/or a light chain variable region sequence ofantibody MED10562.

In some embodiments, the OX40 agonist antibody is an agonist antibodythat binds to the same epitope as any one of the OX40 agonist antibodiesset forth above.

In any of the above embodiments, the OX40 agonist antibody can be afull-length antibody (e.g., IgG1 antibody) or an antibody fragment.

OX40 agonists useful for the methods described herein are in no wayintended to be limited to antibodies. Non-antibody OX40 agonists arecontemplated and well known in the art.

As described above, OX40L (also known as CD134L) serves as a ligand forOX40. As such, agonists that present part or all of OX40L may serve asOX40 agonists. In some embodiments, an OX40 agonist may include one ormore extracellular domains of OX40L. Examples of extracellular domainsof OX40L may include OX40-binding domains. In some embodiments, an OX40agonist may be a soluble form of OX40L that includes one or moreextracellular domains of OX40L but lacks other, insoluble domains of theprotein, e.g., transmembrane domains. In some embodiments, an OX40agonist is a soluble protein that includes one or more extracellulardomains of OX40L able to bind OX40L. In some embodiments, an OX40agonist may be linked to another protein domain, e.g., to increase itseffectiveness, half-life, or other desired characteristics. In someembodiments, an OX40 agonist may include one or more extracellulardomains of OX40L linked to an immunoglobulin Fc domain.

In some embodiments, an OX40 agonist may be an oligomeric or multimericmolecule. For example, an OX40 agonist may contain one or more domains(e.g., a leucine zipper domain) that allows proteins to oligomerize. Insome embodiments, an OX40 agonist may include one or more extracellulardomains of OX40L linked to one or more leucine zipper domains.

In some embodiments, an OX40 agonist may be any one of the OX40 agonistsdescribed in European Patent No. EP0672141 B1.

In some embodiments, an OX40 agonist may be a trimeric OX40L fusionprotein. For example, an OX40 agonist may include one or moreextracellular domains of OX40L linked to an immunoglobulin Fc domain anda trimerization domain (including without limitation an isoleucinezipper domain).

In some embodiments, an OX40 agonist may be any one of the OX40 agonistsdescribed in International Publication No. WO2006/121810, such as anOX40 immunoadhesin. In some embodiments, the OX40 immunoadhesin may be atrimeric OX40-Fc protein. In some embodiments, the OX40 agonist isMEDI6383.

Such combination therapies noted above encompass combinedadministration, whereby the anti-TIGIT antibody and the one or moreagents (e.g., PD-1 axis binding antagonist, OX40 binding agonist, agentthat decreases or inhibits one or more additional immune co-inhibitoryreceptors, and/or additional therapeutic agent) are included in the sameor separate formulations, and separate administration, wherebyadministration of the anti-TIGIT antibody of the invention can occurprior to, simultaneously, and/or following, administration of the one ormore agents (e.g., PD-1 axis binding antagonist, OX40 binding agonist,agent that decreases or inhibits one or more additional immuneco-inhibitory receptors, and/or additional therapeutic agent). In oneembodiment, administration of the anti-TIGIT antibody and administrationof one or more of the agents occur within about one month, or withinabout one, two or three weeks, or within about one, two, three, four,five, or six days, of each other. Anti-TIGIT antibodies of the invention(e.g., 4.1D3 or a variant thereof, e.g., 4.1D3.Q1E) can also be used incombination with radiation therapy.

An anti-TIGIT antibody of the invention (and/or any additionaltherapeutic agent) can be administered by any suitable means, includingparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In some embodiments, the antibody isadministered by subcutaneous administration. In some embodiments, ananti-TIGIT antibody administered by subcutaneous injection exhibits aless toxic response in a patient than the same anti-TIGIT antibodyadministered by intravenous injection. Dosing can be by any suitableroute, for example, by injections, such as intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic. Various dosing schedules including but not limited to single ormultiple administrations over various time-points, bolus administration,and pulse infusion are contemplated herein.

Anti-TIGIT antibodies of the invention would be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of ananti-TIGIT antibody of the invention (e.g., 4.1D3 or a variant thereof,e.g., 4.1D3.Q1E), when used alone or in combination with one or moreother additional agents (e.g., PD-1 axis binding antagonist, OX40binding agonist, agent that decreases or inhibits one or more additionalimmune co-inhibitory receptors, and/or additional therapeutic agent),will depend on the type of disease to be treated, the type of antibodyused, the severity and course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The antibody is suitablyadministered to the patient at one time or over a series of treatments.

As a general proposition, the therapeutically effective amount of theanti-TIGIT antibody administered to human will be in the range of about0.01 to about 100 mg/kg of patient body weight whether by one or moreadministrations. In some embodiments, the antibody used is about 0.01 toabout 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25 mg/kg, about0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 toabout 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1mg/kg administered daily, for example. In one embodiment, an anti-TIGITantibody described herein is administered to a human at a dose of about100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of21-day cycles. The dose may be administered as a single dose or asmultiple doses (e.g., 2 or 3 doses), such as infusions. For repeatedadministrations over several days or longer, depending on the condition,the treatment would generally be sustained until a desired suppressionof disease symptoms occurs. One exemplary dosage of the antibody wouldbe in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one ormore doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or anycombination thereof) may be administered to the patient. Such doses maybe administered intermittently, for example, every week or every threeweeks (e.g., such that the patient receives from about two to abouttwenty, or, for example, about six doses of the anti-TIGIT antibody). Aninitial higher loading dose, followed by one or more lower doses may beadministered. The progress of this therapy is easily monitored byconventional techniques and assays.

In some embodiments, the methods may further comprise an additionaltherapy. The additional therapy may be radiation therapy, surgery,chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy,immunotherapy, bone marrow transplantation, nanotherapy, monoclonalantibody therapy, or a combination of the foregoing. The additionaltherapy may be in the form of adjuvant or neoadjuvant therapy. In someembodiments, the additional therapy is the administration of smallmolecule enzymatic inhibitor or anti-metastatic agent. In someembodiments, the additional therapy is the administration of side-effectlimiting agents (e.g., agents intended to lessen the occurrence and/orseverity of side effects of treatment, such as anti-nausea agents,etc.). In some embodiments, the additional therapy is radiation therapy.In some embodiments, the additional therapy is surgery. In someembodiments, the additional therapy is a combination of radiationtherapy and surgery. In some embodiments, the additional therapy isgamma irradiation. In some embodiments, the additional therapy may be aseparate administration of one or more of the therapeutic agentsdescribed above.

IX. Articles of Manufacture

In another aspect of the invention, an article of manufacture or a kitcontaining materials useful for the treatment, prevention and/ordiagnosis of the disorders described above is provided. The article ofmanufacture comprises a container and a label or package insert on orassociated with the container. Suitable containers include, for example,bottles, vials, syringes, IV solution bags, etc. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is by itself or combined withanother composition effective for treating, preventing and/or diagnosingthe condition and may have a sterile access port (for example thecontainer may be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). At least one active agentin the composition is an anti-TIGIT antibody of the invention. The labelor package insert indicates that the composition is used for treatingthe condition of choice. Moreover, the article of manufacture maycomprise (a) a first container with a composition contained therein,wherein the composition comprises an antibody of the invention; and (b)a second container with a composition contained therein, wherein thecomposition comprises a further cytotoxic or otherwise therapeuticagent. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

In one embodiment, provided is a kit including an anti-TIGIT antibody ofthe invention (e.g., 4.1D3 or a variant thereof, e.g., 4.1D3.Q1E) and apackage insert comprising instructions for using the anti-TIGIT antibodyfor treating or delaying progression of cancer in a subject or fortreating or delaying progression of an immune-related disease in asubject. In a related embodiment, the invention features a kit includingan anti-TIGIT antibody of the invention (e.g., 4.1D3 or a variantthereof, e.g., 4.1D3.Q1E), a PD-1 axis binding antagonist (e.g., a PD-1binding antagonist, a PD-L1 binding antagonist, and a PD-L2 bindingantagonist), and a package insert comprising instructions for using theanti-TIGIT antibody for treating or delaying progression of cancer in asubject or for treating or delaying progression of an immune-relateddisease in a subject. In a related embodiment, the invention features akit including an anti-TIGIT antibody of the invention (e.g., 4.1D3 or avariant thereof, e.g., 4.1D3.Q1E), an OX40 binding agonist (e.g., anOX40 agonist antibody, an OX40L agonist fragment, an OX40 oligomericreceptor, and an OX40 immunoadhesin), and a package insert comprisinginstructions for using the anti-TIGIT antibody for treating or delayingprogression of cancer in a subject or for treating or delayingprogression of an immune-related disease in a subject. In a relatedembodiment, the invention features a kit including an anti-TIGITantibody of the invention (e.g., 4.1D3 or a variant thereof, e.g.,4.1D3.Q1E), a PD-1 axis binding antagonist (e.g., a PD-1 bindingantagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist),an OX40 binding agonist (e.g., an OX40 agonist antibody, an OX40Lagonist fragment, an OX40 oligomeric receptor, and an OX40immunoadhesin), and a package insert comprising instructions for usingthe anti-TIGIT antibody for treating or delaying progression of cancerin a subject or for treating or delaying progression of animmune-related disease in a subject. In any of the above embodiments,the subject may, for example, be a human. It is specificallycontemplated that any of the anti-TIGIT antibodies, OX40 bindingagonists, and PD-1 axis binding antagonists described herein may beincluded in the kit.

In yet another embodiment, provided is a kit including an anti-TIGITantibody of the invention (e.g., 4.1D3 or a variant thereof, e.g.,4.1D3.Q1E) and a package insert comprising instructions for increasing,enhancing, or stimulating an immune response or function in a subject.In a related embodiment, the invention features a kit including ananti-TIGIT antibody of the invention (e.g., 4.1D3 or a variant thereof,e.g., 4.1D3.Q1E), a PD-1 axis binding antagonist (e.g., a PD-1 bindingantagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist),and a package insert comprising instructions for increasing, enhancing,or stimulating an immune response or function in a subject. In a relatedembodiment, the invention features a kit including an anti-TIGITantibody of the invention (e.g., 4.1D3 or a variant thereof, e.g.,4.1D3.Q1E), an OX40 binding agonist (e.g., an OX40 agonist antibody, anOX40L agonist fragment, an OX40 oligomeric receptor, and an OX40immunoadhesin), and a package insert comprising instructions for usingthe anti-TIGIT antibody for increasing, enhancing, or stimulating animmune response or function in a subject. In a related embodiment, theinvention features a kit including an anti-TIGIT antibody of theinvention (e.g., 4.1D3 or a variant thereof, e.g., 4.1D3.Q1E), a PD-1axis binding antagonist (e.g., a PD-1 binding antagonist, a PD-L1binding antagonist, and a PD-L2 binding antagonist), an OX40 bindingagonist (e.g., an OX40 agonist antibody, an OX40L agonist fragment, anOX40 oligomeric receptor, and an OX40 immunoadhesin), and a packageinsert comprising instructions for using the anti-TIGIT antibody forincreasing, enhancing, or stimulating an immune response or function ina subject. In any of the above embodiments, the subject may, forexample, be a human. It is specifically contemplated that any of theanti-TIGIT antibodies, OX40 binding agonists, and PD-1 axis bindingantagonists described herein may be included in the kit.

Examples

The invention can be further understood by reference to the followingexamples, which are provided by way of illustration and are not meant tobe limiting.

Example 1. Generation of Anti-TIGIT Antibodies

Either Sprague Dawley rats or OMT transgenic rats (Open MonoclonalTechnology, Palo Alto, Calif.) were co-immunized with an initial dosesubcutaneously of 50 μg human TIGIT protein (Genentech, Inc.) and 50 μgcynomolgus monkey (cyno) TIGIT protein (Genentech, Inc.) mixed withComplete Freund's Adjuvant (BD, Franklin Lakes, N.J.), followed by 25 μghuman TIGIT protein and 25 μg cyno TIGIT protein diluted in PBS inmultiple sites subcutaneously and intraperitoneally every two weeks.

Multiple lymph nodes were harvested three days after the lastimmunization. B cells from these rats were enriched using negativeselection with biotinlyated antibodies targeting rat non-B cells (BD;eBioscience, San Diego, Calif.) and streptavidin-coated magnetic beads(Miltenyi, San Diego, Calif.). The resulting B cell population was fusedwith P3X63-Ag8U.1 mouse myeloma cells (American Type Culture Collection,Rockville, Md.) via electrofusion (Harvard Apparatus, Holliston, Mass.).Fused cells were incubated at 37° C., 7% CO₂, overnight in Medium C(StemCell Technologies, Vancouver, BC, Canada), before resuspension insemi-solid Medium D (StemCell Technologies) with anti-rat IgG-FITC(Sigma-Aldrich, St. Louis, Mo.) and plating into Omniwell trays (ThermoFisher Scientific, Rochester, N.Y.). Seven days after plating,fluorescent colonies were selected and transferred into 96-well cultureplates (BD) containing Medium E (StemCell Technologies) using a ClonepixFL (Molecular Devices, Sunnyvale, Calif.). Supernatants were screened byELISA against human TIGIT protein seven days after colony picking.384-well plates (Greiner Microlon, Greiner Bio-One, Monroe, N.C.) werecoated with 1 μg/ml of protein diluted in 0.05 M sodium carbonatebuffer, pH 9.6, and incubated overnight at 4° C. Then wells were blockedwith 100 μl of PBS containing 0.5% BSA and 0.5% Tween-20 for one hour atroom temperature. After washing, supernatants and/or sera were added at50 μl and shaken for 30 minutes at room temperature. For detection ofspecific antibodies, horseradish peroxidase-conjugated anti-rat IgGantibodies (Bethyl Laboratories, Montgomery, Tex.) were diluted to anoptimized concentration in PBS containing 0.5% BSA and 0.5% Tween-20 andadded at 50 μl per well after washing, and plates were shaken for 30minutes at room temperature. Plates were washed three times with 100 μlPBS containing 0.05% Tween-20. 50 μl TMB Substrate (BioFX, Owings Mills,Md.) was added and plates were incubated for five minutes at roomtemperature, followed by the addition of 50 μl stop solution (BioFX),then read at 630 nm. Human TIGIT-binding hybridoma cell lines wereexpanded and cultured for two to four days, then screened by ELISAagainst human TIGIT, cyno TIGIT, and mouse TIGIT proteins (Genentech,Inc.). Supernatant from human and cyno cross-reactive cell lines washarvested and purified by protein G (Protein G Sepharose 4 Fast Flow, GEHealthcare, Pittsburgh, Pa.). Approximately 500 clones were screened.From the screening, clones 4.1D3, 7.4A3, and 4.1A4 were identified fromthe immunization of OMT rats, and clones 1.662, 1.10A5, 1.7E7, and1.15C8 were identified from the immunization of Sprague Dawley rats.

Example 2. Optimization of Anti-TIGIT Antibodies

A. OMT Rat-Derived Anti-TIGIT Human Monoclonal Antibody Polishing

To prevent unwanted pyroglutamate formation for clones 4.1A4, 4.1D3, and7.4A3 (also referred to as 1A4, 1D3, and 4A3, respectively) and toresolve the issue of unpaired cysteine residues for clones 4.1A4 and7.4A3, the following optimized anti-TIGIT antibody variants weregenerated: 4.1A4.C96S.Q1E, 4.1A4.C96Y.Q1E, 4.1D3.Q1E, 7.4A3.C96S.Q1E,and 7.4A3.C96Y.Q1E (also referred to as 1A4.C96S.Q1E, 1A4.C96Y.Q1E,1D3.Q1E, 4A3.C96S.Q1E, and 4A3.C96Y.Q1E, respectively). As describedbelow in Table 2, the affinities of these five optimized variants forhuman and cynomolgus monkey (cyno) TIGIT were then determined by SurfacePlasmon Resonance (SRP) (BIACORE™ analysis) and compared to that oftheir respective parental clones.

Briefly, a series S CM5 biosensor chip was activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) reagents according to the supplier's (GEHealthcare Biosciences, Piscataway, N.J.) instructions, and a humanantibody capture kit was applied to couple goat anti-human Fc IgGs inorder to achieve approximately 10,000 response units (RU) on each flowcell, followed by blocking un-reacted groups with 1M ethanolamine.

For kinetics measurements, each clone was captured to achieveapproximately 250 RU and 5-fold serial dilutions of TIGIT antigen (1.23nM to 300 nM) were injected in HBS-P buffer (0.01 M HEPES pH 7.4, 0.15MNaCl, 0.005% surfactant P20) at 25° C. (flow rate: 30 μl/min) with noregeneration between injections. The sensorgrams were recorded andevaluated by BIAcore™ T200 Evaluation Software (version 2.0) aftersubtraction of reference cell signal. Association rates (k_(on)) anddissociation rates (k_(off)) were calculated using a simple one-to-oneLangmuir binding model. The equilibrium dissociation constant (K_(D))was calculated as the ratio k_(off)/k_(on).

TABLE 2 BIACORE ™ affinity measurements for OMT rat-derived 1A4, 1D3,and 4A3 antibody variants against human and cyno TIGIT Human TIGIT CynoTIGIT Kon Koff KD Kon Koff KD xTIGIT mAbs (10⁵ M⁻¹s⁻¹) (10⁻⁴ s⁻¹) (nM)(10⁵ M⁻¹s⁻¹) (10⁻⁴ s⁻¹) (nM) 1A4 3.42 12.2 3.57 2.66 6.43 2.421A4.C96S.Q1E 12.5 367.9 29.4 13.4 169.1 12.6 1A4.C96Y.Q1E — — >100 —— >100 1D3 38.4 16.4 0.43 6.21 20.7 3.33 1D3.Q1E 17.2 9.7 0.56 4.78 15.43.22 4A3 3.02 10.4 3.44 3.34 6.99 2.09 4A3.C96S.Q1E 2.88 8.56 2.97 2.765.56 2.01 4A3.C96Y.Q1E 3.72 364.7 98.0 3.95 140.5 35.6

The clones 4.1A4.C96S.Q1E, 7.4A3.C96S.Q1E, and 4.1D3.Q1E were consideredas final molecules with similar and acceptable binding affinity to bothhuman and cyno TIGIT by comparison with the parental clones. AdditionalBIACORE™ analyses revealed that the 4.1D3.Q1E clone, in particular, wasable to cross-react and bind to rabbit TIGIT with low nanomolar affinity(Table 3).

TABLE 3 4.1D3.Q1E is capable of cross-reacting with human, cyno, andrabbit TIGIT BIAcore Kinetics Analysis Human Cyno Rabbit TIGIT TIGITTIGIT Source Clone Monovalent Affinity (nM) OMT 4.1D3.Q1E 0.56 3.2 4.97.4A3.Q1E.C96S 2.97 2.01 >500 Mouse h1A5.L2H8 5.76 >500 >500

B. Humanization of SD Rat-Derived Anti-TIGIT Monoclonal Antibodies

Monoclonal antibodies 1.662, 1.10A5, 1.7E7, and 1.15C8 (as referred toherein as 662, 10A5, 7E7, and 15C8, respectively) were humanized asdescribed below. Residue numbers are according to Kabat et al.(Sequences of Proteins of Immunological Interest. 5th Ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991)).

For 6B2 antibody humanization, hypervariable regions from the rat 6B2(rat6B2) antibody were engineered into its closest human acceptorframeworks to generate humanized 6B2 (h6B2.L1H1). Specifically, from therat6B2 VL domain, positions 24-34 (L1), 50-56 (L2), and 89-97 (L3) weregrafted into human germline IGKV1-33*01. From the rat6B2 VH domain,positions 26-35 (H1), 50-65 (H2), and 95-102 (H3) were grafted intohuman germline IGHV2-5*08. In addition, position 43 in framework II ofVL and positions 69, 73, 78 in framework III of VH were retained fromthe rat sequence in h6B2.L1H1. Those residues were found to be part ofthe framework residues acting as “Vernier” zone, which may adjustCDR/HVR structure and fine-tune the antigen fit. See, e.g., Foote andWinter, J. Mol. Biol. 224: 487-499 (1992). These CDR/HVR definitionsinclude positions defined by their sequence hypervariability (Wu, T. T.& Kabat, E. A. (1970)), their structural location (Chothia, C. & Lesk,A. M. (1987)) and their involvement in antigen-antibody contacts(MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)).

Additional humanized 6B2 variants were generated by engineering each ratresidue of h6B2.L1H1 at Vernier zone back to human germline residue andthese variants are h6B2.L2H1 (VL: S43A), h6B2.L1H2 (VH: V69I), h6B2.L1H3(VH: A73T), h6B2.L1H4 (VH: A78V), h6B2.L1H5 (VH: V69I, A73T, A78V), andh6B2.L2H5 (VL: S43A, VH: V69I, A73T, A78V; a fully humanized version).

For 10A5 antibody humanization, hypervariable regions from the rat 10A5(rat10A5) antibody were engineered into its closest human acceptorframeworks to generate humanized 10A5 (h10A511H1). Specifically, fromthe rat10A5 VL domain, positions 24-34 (L1), 50-56 (L2), and 89-97 (L3)were grafted into human germline IGKV3-15*01. From the rat10A5 VHdomain, positions 26-35 (H1), 50-65 (H2) and 95-102 (H3) were graftedinto human germline IGHV2-5*08. In addition, position 43 in framework IIand position 58 in framework III of VL and positions 69 and 78 inframework III of VH were retained from the rat sequence in h10A5.L1H1.Those residues were found to be part of the framework residues acting as“Vernier” zone as described above.

Additional humanized 10A5 variants were generated by engineering eachrat residue of h10A5L1H1 at Vernier zone back to human germline residueand these variants are h10A5.L2H1 (VL: S43A), h10A5.L3H1 (VL: V581),h10A5.L4H1 (VL: S43A, V581), h10A5.L1H2 (VH: V69I), h10A5.L1H3 (VH:A78V), h10A5.L1H4 (VH: V69I, A78V), and h10A5.L4H4 (VL: S43A, V581, VH:V69I, A78V; a fully humanized version)

For 7E7 antibody humanization, hypervariable regions from the rat 7E7(rat7E7) antibody were engineered into its closest human acceptorframeworks to generate humanized 7E7 (h7E7.L1H1). Specifically, from therat7E7 VL domain, positions 24-34 (L1), 50-56 (L2), and 89-97 (L3) weregrafted into human germline IGKV1-9*01. From the rat7E7 VH domain,positions 26-35 (H1), 50-65 (H2), and 95-102 (H3) were grafted intohuman germline IGHV1-3*01. In addition, positions 58, 71, and 87 inframework III of VL, positions 47, 48 in framework II of VH andpositions 67, 69, 71, 91, and 93 in framework III of VH were retainedfrom the rat sequence in h7E7.L1H1. Those residues were found to be partof the framework residues acting as “Vernier” zone as described above.

Additional humanized 7E7 variants were generated by engineering each ratresidue of h7E7.L1H1 at Vernier zone back to human germline residue andthese variants are h7E7.L2H1 (VL: I58V), h7E7.L3H1 (VL: Y71F), h7E7.L4H1(VL: F87Y), h7E7.L5H1 (VL: I58V, Y71F, F87Y), h7E7.L1H2 (VH: 147W),h7E7.L1H3 (VH: I48M), h7E7.L1H4 (VH: A67V), h7E7.L1H5 (VH: L69I),h7E7.L1H6 (VH: A71R), h7E7.L1H7 (VH: F91Y), h7E7.L1H8 (VH: T93A),h7E7.L1H9 (VH: 147W, I48M, A67V, L69I, A71R, F91Y, T93A), and h7E7.L5H9(VL: I58V, Y71F, F87Y, VH: 147W, I48M, A67V, L69I, A71R, F91Y, T93A; afully humanized version).

For 15C8 antibody humanization, hypervariable regions from the rat 15C8(rat15C8) antibody were engineered into its closest human acceptorframeworks to generate humanized 15C8 (h15C8.L1H1). Specifically, fromthe rat1508 VL domain, positions 24-34 (L1), 50-56 (L2), and 89-97 (L3)were grafted into human germline IGKV1-9*01. From the rat1508 VH domain,positions 26-35 (H1), 50-65 (H2), and 95-102 (H3) were grafted intohuman germline IGHV1-3*01. In addition, positions 58, 71, and 87 inframework III of VL, positions 37, 47, and 48 in framework II of VH, andpositions 67, 69, 71, and 91 in framework III of VH were retained fromthe rat sequence in h15C8.L1H1. Those residues were found to be part ofthe framework residues acting as “Vernier” zone as described above.

Additional humanized 15C8 variants were generated by engineering eachrat residue of h15C8.L1H1 at Vernier zone back to human germline residueand these variants are h15C8.L2H1 (VL: I58V), h15C8.L3H1 (VL: Y71F),h15C8.L4H1 (VL: F87Y), h15C8.L5H1 (VL: I58V, Y71F, F87Y), h15C8.L1H2(VH: L37V), h15C8.L1H3 (VH: 147W), h15C8.L1H4 (VH: I48M), h15C8.L1H5(VH: A67V), h15C8.L1H6 (VH: L69I), h15C8.L1H7 (VH: T71R), h15C8.L1H8(VH: F91Y), h15C8.L1H9 (VH: L37V, 147W, I48M, A67V, L69I, T71R, F91 Y),and h7E7.L5H9 (VL: I58V, Y71F, F87Y, VH: L37V, 147W, I48M, A67V, L69I,T71R, F91 Y; a fully humanized version).

The first humanized versions, h6B2, L1H1, h10A5.L1H1, h7E7, L1H1, andh15C8.L1H1, which contain both rat CDRs and framework residues atVernier positions, and the fully humanized versions, h6B2.L2H5,h10A5.L4H4, h7E7.L5H9, and h15C8.L5H9, which contain only rat CDRs weresubject to BIAcore affinity measurement by comparison with original ratantibodies, rat6B2, rat10A5, rat7E7 and rat 15C8. For binding affinitydeterminations of TIGIT antibodies against human TIGIT, cynomolgusmonkey TIGIT and human alanine-scanned TIGIT mutants by single-cyclekinetics, Surface Plasmon Resonance (SRP) measurement with aBIACORE™-T200 instrument was used, as described above. The BIACORE™results in Table 4, below, indicate that all the humanized variantsexhibit a drop in binding affinity (i.e., an increase in K_(D)) of about5- to 10-fold against both human and cyno TIGIT. The fully humanizedversions of h6B2.L2H5 and h10A5.L4H4 surprisingly show similar bindingaffinity as the first humanized versions of h6B2.L1H1 and h10A5.L1H1,suggesting that rat6B2 and rat10A5 framework residues at Vernierpositions do not contribute to human and cyno TIGIT binding. Therefore,both h6B2.L2H5 and h10A5.L4H4 represent the final humanized version forrat6B2 and rat10A5, respectively. By contrast, the fully humanizedversions of h7E7.L5H9 and h15C8.L5H9 lost the ability to bind human andcyno TIGIT.

To further elucidate which rat framework residues at Vernier positionsfor the first humanized antibodies h7E7, L1H1, and h15C8.L1H1 arecrucial for binding, all the framework polishing variants were subjectto BIAcore affinity measurement. For 7E7 framework polishing, variantsof h7E7.L1H2, h7E7.L1H9, and h7E7.L5H9 containing VH: 147W substitutionshow human and cyno TIGIT binding affinity drop more than 100-fold, andvariants of h7E7.L3H1 and h7E7.L5H1, containing VL: Y71F substitution,show cyno TIGIT binding affinity drop about 2-3 fold. Therefore, bothVH: 147 and VL: Y71 rat framework residues were kept in the final 7E7humanized version, called h7E7.L5aH9a, to retain human and cyno TIGITbinding. For 15C8 framework polishing, variants of h15C8.L1H3,h15C8.L1H9, and h15C8.L5H9, containing VH: 147W, substitution show humanand cyno TIGIT binding affinity drop more than 100-fold, and variants ofh15C8.L3H1 and h15C8.L5H1 containing VL: Y71F substitution show humanand cyno TIGIT binding affinity drop about 2-3 fold. Other variants,h15C8.L1H2, h15C8.L1H5, and h15C8.L1H7, contain VH: L37V, A67V, and T71Rsubstitution, respectively, also show human TIGIT binding affinity dropabout 2-3 fold. Therefore, all five VH: 147, L37, A67, T71 and VL: Y71rat framework residues were kept in the final 15C8 humanized version,called h15C8.L5aH9a, to retain human and cyno TIGIT binding.

TABLE 4 BIACORE ™ affinity measurements summary for humanized SDrat-derived 6B2, 10A5, 7E7, and 15C8 antibody variants against human andcyno TIGIT Human TIGIT Cyno TIGIT Kon Koff KD Kon Koff KD xTIGIT mAbs(10⁵ M⁻¹s⁻¹) (10⁻⁴ s⁻¹) (nM) (10⁵ M⁻¹s⁻¹) (10⁻⁴ s⁻¹) (nM) rat6B2 9.970.35 0.04 6.88 2.02 0.29 h6B2.L1H1 46.7 25.3 0.54 12.3 11.8 0.96h6B2.L2H5 46.5 22.2 0.48 12.8 12.4 0.97 rat10A5 9.05 0.84 0.09 6.78 2.810.41 h10A5.L1H1 45.7 23.8 0.52 14.7 12.7 0.86 h10A5.L4H4 52.1 22.7 0.4415.2 12.9 0.85 rat7E7 5.68 0.18 0.03 3.99 0.27 0.07 h7E7.L1H1 20.6 4.760.23 4.18 1.52 0.36 h7E7.L2H1 20.6 5.04 0.24 4.93 1.79 0.36 h7E7.L3H19.2 2.18 0.24 12.8 12.2 0.95 h7E7.L4H1 29.5 7.96 0.27 6.29 2.74 0.44h7E7.L5H1 32.1 10.4 0.32 5.75 3.24 0.56 h7E7.L1H2 — — >100 — — >100h7E7.L1H3 28.2 10.7 0.38 9.08 3.81 0.42 h7E7.L1H4 27.9 7.49 0.27 6.012.43 0.40 h7E7.L1H5 32.4 12.9 0.40 8.98 3.59 0.40 h7E7.L1H6 18.5 6.480.35 6.41 2.84 0.44 h7E7.L1H7 27.4 8.49 0.31 6.64 2.86 0.43 h7E7.L1H827.8 10.1 0.36 8.45 3.31 0.39 h7E7.L1H9 — — >100 — — >100 h7E7.L5H9 —— >100 — — >100 h7E7.L5aH9a 12.8 3.63 0.28 5.91 1.82 0.31 rat15C8 5.220.18 0.03 3.81 0.96 0.25 h15C8.L1H1 9.22 3.57 0.39 4.93 4.64 0.94h15C8.L2H1 7.71 5.06 0.66 5.02 5.02 1.00 h15C8.L3H1 6.31 5.33 0.84 3.365.88 1.75 h15C8.L4H1 9.55 6.11 0.64 4.85 5.59 1.15 h15C8.L5H1 6.74 5.620.83 2.81 7.52 2.68 h15C8.L1H2 10.4 10.7 1.03 3.82 8.06 2.1 h15C8.L1H3 —— >100 — — >100 h15C8.L1H4 8.41 4.98 0.59 5.32 5.58 1.05 h15C8.L1H5 7.985.67 0.71 4.84 5.58 1.15 h15C8.L1H6 13.8 7.56 0.55 6.63 5.56 0.84h15C8.L1H7 6.1 5.97 0.98 2.19 2.06 0.94 h15C8.L1H8 6.2 3.81 0.61 2.911.19 0.41 h15C8.L1H9 — — >100 — — >100 h15C8.L5H9 — — >100 — — >100h15C8.L5aH9a 11.2 4.21 0.38 5.96 6.13 1.03

Example 3. In Vitro Binding and Blocking Studies of Anti-TIGITAntibodies

A. In Vitro Binding Characterization of Anti-TIGIT Antibodies

The OMT- and SD-derived anti-TIGIT antibodies were characterized invitro for their ability to bind to TIGIT and to block poliovirusreceptor (PVR) binding to TIGIT. Binding to human and cyno TIGIT wastested by BIACORE™ analysis, as described above, in the context of bothmonovalent and bivalent affinity, and compared to a mouse-derivedanti-TIGIT antibody (1A5) and a chimeric hamster-derived anti-TIGITantibody (10A7; see, e.g., U.S. Pub. No. 2009/0258013, which isincorporated herein by reference in its entirety). The results of theBIACORE™ analysis, depicted in Table 5, indicate that numerousOMT-derived anti-TIGIT clone variants, such as 4.1D3.Q1E and7.4A3.C96S.Q1E, and SD-derived anti-TIGIT clone variants were capable ofbinding to both human and cyno TIGIT, each with a high and similaraffinity. Such properties are desirable for therapeutic anti-TIGITantibodies as they allow for facile toxicity testing using cynomolgusmonkey (Macaca fascicularis) as a non-clinical toxicology species.Additional cross-reactivity of the 4.1D3.Q1E clone with rabbit mayprovide an additional toxicology species.

TABLE 5 BIACORE ™ analysis of optimized OMT- and SD-derived anti-TIGITclones BIAcore Kinetics Analysis Human TIGIT Cyno TIGIT Human TIGIT CynoTIGIT Source Clone Monovalent Affinity (nM) Bivalent Affinity (nM) OMTRat 4.1A4.Q1.C96 4.9 2.6 0.31 0.035 4.1A4.Q1E.C96 3.6 2.4 4.1A4.Q1E.C96S29.4 12.6 4.1A4.Q1E.C96Y >100 >100 4.1D3.Q1 0.61 3.4 ≤0.019 0.364.1D3.Q1E 0.57 3.2 7.4A3.Q1.C96 3.60 1.6 0.098 ≤0.05 7.4A3.Q1E.C96 3.452.09 7.4A3.Q1E.C96S 2.97 2.02 7.4A3.Q1E.C96Y 98.1 35.5 SD Rat Chimeric1.6B2 0.035 0.29 ≤0.027 0.091 h1.6B2.L1H1 0.54 0.96 Chimeric 1.10A50.093 0.41 ≤0.003 0.013 h1.10A5.L1H1 0.52 0.86 Chimeric 1.7E7 ≤0.0180.067 ≤0.004 ≤0.009 h1.7E7.L1H1 0.33 0.51 Chimeric 1.15C8 0.034 0.25≤0.007 ≤0.013 h1.15C8.L1H1 0.58 1.39 Mouse h1A5.L2H8 5.76 >500 0.26 7.77Hamster c10A7 (to human) 24 0.54 8.22 c10A7 (to murine) 0.24

Additional in vitro binding studies of the OMT- and SD-derivedanti-TIGIT antibody clones were conducted. The OMT- and SD-derivedanti-TIGIT clones were tested for binding to human and cyno TIGITexpressed on the surface of CHO cells by FACS analysis. CHO cells weretransfected with full-length human TIGIT (CHO-hTIGIT) or cynomologousTIGIT (CHO-cyTIGIT). The TIGIT-expressing CHO cells were incubated witheach anti-TIGIT antibody at the indicated concentrations and thendetected via a FITC-labeled anti-hIgG antibody (see FIG. 1A) usingstandard methods. The results, shown in Table 6 and FIGS. 1B-1E,corroborate the results from the BIACORE™ analysis, described above.

The OMT- and SD-derived anti-TIGIT clones were also tested for bindingto human primary T cells by FACS analysis. Human PBMC were activated forwith plate-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (2 μg/ml) for2 days as described in Yu et al. Nature Immunology. 10: 48-57, 2009,which is incorporated herein by reference in its entirety. Cells werethen washed and incubated with incubated with each anti-TIGIT antibodyat the indicated concentrations and then detected via a FITC-labeledanti-h IgG antibody. Cells were also stained with anti-CD4, anti-CD8,and anti-CD25 (BD bioscience). Human CD4+ (CD25+CD4+) and human CD8+(CD25+CD8+) T cells were assessed for binding by the anti-TIGIT clonesby FACS. All samples were acquired on LSR-II or LSR-Fortessa instruments(BD Biosciences) and analyzed using FlowJo software (Treestar). Asdescribed in Table 6 and FIGS. 2A-2D, the clones exhibited approximatelyequivalent binding to their target TIGIT.

TABLE 6 Summary of CHO-TIGIT and human T cell binding of optimized OMT-and SD-derived anti-TIGIT clones 10A7 (mu TIGIT) & OMT clones SD clonesAssay 1A5 (hu TIGIT) (hu TIGIT) (hu TIGIT) CHO-TIGIT 10A7 0.54 nM4.1A4.C96S.Q1E 0.90 nM 1.6B2 binding  1A5 0.14 nM 4.1D3.Q1E 1.04 nM 0.52nM (EC50) 7.4A3.C96S.Q1E 3.04 nM 1.7E7 0.10 nM Human T cell 10A7 0.63 nM4.1A4.C96S.Q1E 0.38 nM 1.6B2 binding  1A5 0.14 nM 4.1D3.Q1E 1.36 nM 0.09nM (EC50) 7.4A3.C96S.Q1E 2.63 nM 1.7E7 0.11 nM

B. In Vitro Blocking Characterization of Anti-TIGIT Antibodies

The OMT- and SD-derived anti-TIGIT antibody clones were also tested fortheir ability to block TIGIT-PVR and TIGIT-CD226 interactions.

To test for the ability of the clones to block the TIGIT-PVRinteraction, a blocking ELISA assay was used. Briefly, recombinant humanPVR-Fc fusion protein, cynomolgus monkey PVR-Fc fusion protein, or mousePVR-Fc fusion protein was coated at 4 μg/ml at 25 μl/well inphosphate-buffered saline (PBS, pH 7.0) onto 384-well Maxisorpmicrotiter plates (NUNC, Denmark) and incubated overnight at 4° C. Thecoating solution was then discarded and the plate was blocked using 0.5%bovine serum albumin (BSA) in PBS at 80 μl/well and incubated withgentle agitation for 1 hour. Titration curves of anti-TIGIT and Fabisotype control antibodies were diluted in assay buffer (PBS, 0.5% BSA,0.05% Tween 20) using 12 serial 2.5-fold dilutions (40000-1.7 ng/mL).Equal volumes of either human TIGIT-AviFlag-biotin (1 μg/mL), cynomolgusTIGIT-AviFlag-biotin (150 ng/mL), or mouse TIGIT-His (1 μg/mL) wereadded to these titration curves and incubated at room temperature for 1hour with gentle agitation. The blocked plate was washed 3× with washbuffer (PBS, 0.05% Tween 20, pH 7.4) using an ELx405 automatedmicrotiter plate washer (BioTek Instruments, Vermont) and theantibody/ligand-biotin or ligand-His mixture was added at 25 μl/well andincubated for 1 hour at room temperature with gentle agitation. Theplate was then washed 6× with wash buffer and bound humanTIGIT-AviFlag-biotin, cyno TIGIT-AviFlag-biotin, or mouse TIGIT-His wasdetected by adding 25 μl/well of horseradish peroxidase-conjugatedstreptavidin (1:10000, Amersham, UK) or horseradishperoxidase-conjugated mouse anti-His (1:2500, Qiagen, Germany) dilutedin assay buffer. After a 30 minute or 1 hour incubation, the plate waswashed 6× with wash buffer and 100 μl/well of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (KPL, Inc., Gaithersburg, Md. USA) was addedto allow color to develop. The reaction was quenched by addition of 1 Mphosphoric acid. Absorbance was read at 450 nm on a MULTISKAN ASCENT®microtiter plate reader (Thermo Labsystems, Helsinki, Finland). Fitcurves were plotted from the resulting optical density values from theELISA plates and IC50s were calculated using Genedata Screener software(Genedata, Switzerland). As shown below in FIGS. 3A-3D, these assaysdemonstrated the cross-reactivity and blocking ability of 4.1D3.Q1E forhuman TIGIT and cyno TIGIT, which distinguishes 4.1D3.Q1E from thehamster-derived anti-TIGIT antibody, 10A7, which cross-reacts with mouseTIGIT, but not cyno TIGIT. The data indicate that 4.1D3.Q1E stronglyblocks the binding of cyno TIGIT to cyno PVR. The data also indicatethat 4.1D3.Q1E is a stronger blocker of human TIGIT to human PVR thanthe 10A7 hamster-derived anti-TIGIT antibody clone.

These studies were further validated in CHO-TIGIT PVR blockingexperiments in which TIGIT-expressing CHO cells were incubated withlabeled PVR-Fc fusion protein in the presence of anti-TIGIT antibody oran human IgG1 control (FIG. 4A). As shown in Table 7 and FIGS. 4B-4E,incubation with 10 μg/ml of OMT-derived anti-TIGIT antibody (FIGS. 4Band 4C) or SD-derived anti-TIGIT antibody (FIGS. 4D and 4E) resulted inblocking of TIGIT-PVR binding for both huTIGIT-huPVR and cyTIGIT-cyPVR,with IC50 values in the nanomolar and subnanomolar range.

To test for the ability of the clones to block the TIGIT-CD226interaction, TR-FRET (Time-resolved Fluorescence Resonance EnergyTransfer) was used. First, human SNAP-tagged (ST) CD226 withnon-permeant donor and acceptor fluorophores was expressed and labeledusing CHO cells. The human ST-CD226 was co-expressed with HA-taggedTIGIT, in the presence or the absence of the OMT- and SD-derivedanti-TIGIT antibody clones. The addition of each of the tested clones tothe cell cultures significantly reduced the ability of TIGIT and CD226to associate (Table 7). These data suggested that anti-TIGIT treatmentwith the identified anti-TIGIT antibodies can limit TIGIT's interactionwith CD226, indicating that these antibodies may be favorabletherapeutics based on their ability to activate T cell activity byreleasing TIGIT-mediated suppression of CD226 activity and preventingsubsequent CD8+ T cell exhaustion.

TABLE 7 Summary of TIGIT-PVR blocking and TIGIT-CD226 blocking ofoptimized OMT- and SD- derived anti-TIGIT clones 10A7 (mu TIGIT) & 1A5OMT clones SD clones Assay (hu TIGIT) (hu TIGIT) (hu TIGIT) TIGIT-PVR10A7 0.07 nM 4.1A4.C96S.Q1E 0.14 nM 1.662 blocking  1A5 0.18 nM4.1D3.Q1E 0.29 nM 0.08 nM (IC50) 7.4A3.C96S.Q1E 0.38 nM 1.7E7 0.04 nMTIGIT-CD226 10A7 17.6 nM 4.1A4.C96S.Q1E 25.2 nM FRET (IC50)  1A5 13.7 nM4.1D3.Q1E 45.0 nM 7.4A3.C96S.Q1E 19.3 nM

Example 4. Pharmacokinetic Characterization of Anti-TIGIT Antibodies

Next, the pharmacokinetic (PK) properties of OMT- and SD-derivedanti-TIGIT antibody clones were tested in cynomolgus monkeys as comparedto the mouse-derived anti-TIGIT antibody bearing an N297G aglycosylationmutation (1A5 N297G) and an anti-gD control antibody. In theseexperiments, serum concentration of the antibodies was measured over thespan of 28 days following 10 mg/kg intravenous administrations incynomolgus monkeys. The PK studies revealed that the SD-derived h1.662variant exhibited the fastest clearance, at approximately 20 ml/day/kg(FIGS. 5A and 5B). The tested 1A5 variant also exhibited fast clearance,at approximately 15 ml/day/kg (FIGS. 5A and 5B). In contrast to theseanti-TIGIT variants, the tested OMT variants (4.1D3.Q1E, 7.4A3.C96S.Q1E,and 4.1A4.C96S.Q1E) exhibited a clearance of approximately 8-10ml/day/kg (FIGS. 5A and 5B), which is on the high end of thehistorically accepted cyno range of approximately 4-8 ml/day/kg. The PKprofiles of the OMT variants were similar to the control anti-gDantibody through dl 0, at which time target-mediated drug disposition(TMDD) and/or anti-therapeutic antibody (ATA) effects convolute theestimates.

The OMT-derived anti-TIGIT antibody 4.1D3, which exhibited favorable PKproperties, was also compared to humanized 10A7 (h10A7.K4G3) in a 7-dayPK study. In this experiment, eight female cynomolgus monkeys werebinned into two groups of four monkeys, with the first group receivingh10A7.K4G3 and the second group receiving 4.1D3. Both anti-TIGITantibodies were administered at a dose level of 10 mg/kg (dose volume of5 ml/kg; dose concentration of 2 mg/ml) by slow bolus intravenousinjection. Blood samples (0.5 ml) were collected at 0 hour (pre-dose),0.25 hour, 2 hours, 8 hours, 1 day, 3 days, and 7 days post-dose inaccordance with the following collection windows:

Postdose Time Point Collection Window  0 to 15 minutes +/− 1 minute  16to 30 minutes +/− 2 minutes 31 to 45 minutes +/− 3 minutes 46 to 60minutes +/− 4 minutes 61 minutes to 2 hours +/− 5 minutes  >2 to 8 hours+/− 10 minutes  >8 to 24 hours +/− 20 minutes using gel serum separator tubes. The blood was maintained at ambienttemperature and allowed to clot for at least 20 minutes prior tocentrifugation. Samples were centrifuged (approximately 10 to 15 minutesat approximately 1500 to 2000×g [force], 2° C. to 8° C.) within 1 hourof collection. Serum was harvested within 30 minutes of the start ofcentrifugation and transferred into 0.5-mL 2D bar-coded screw top tubes(Thermofisher Scientific catalog #3744 or equivalent). The serum samplewas labeled with the animal number, species, dose group, day ofcollection, sample type (i.e., PK-serum), and study numbers. The serumsamples were then analyzed for concentrations of h10A7.K4G3 or 4.1D3. Asshown in FIGS. 5C and 5D, this PK study revealed that h10A7.K4G3exhibited a faster clearance (lower AUC) than 4.1D3, and that theclearance of h10A7.K4G3 (>9 ml/day/kg) is faster than recommended formonoclonal antibody selection in cynomolgus monkey. The estimatedclearance was based on data from day 0 through day 7 and calculated byWinnonlin using NCA.

Example 5. Molecular Analysis of Anti-TIGIT Antibodies

OMT-derived (4.1D3, 7.4A3.C96S, and 4.1A4.C96S) and SD-derived(h1.6132.L1H1, h1.10A5.L1H1, h1.7E7.L1H1, and h1.15C8.L1H1) anti-TIGITantibody clones were also tested in molecular assessment (MA) analysesfor stable cell line properties. Briefly, the anti-TIGIT antibodies (1mg/ml) were tested for stress under chemical conditions with AAPH(2,2-azobis(2-amidinopropane) dihydrochloride), a small molecule knownto generate free radicals, as well as under thermal conditions atvarying pH (a two-week thermal stress test at 40° C., at either pH 5.5or pH 7.4). As shown in Tables 8 and 9, below, 4.1A4.0965 was determinedto be unstable, showing unacceptable main peak loss and increaseddeamidation in the MA studies. Similarly, h1.7E7.L1H1 and h1.15C8.L1H1showed high initial deamidation (>30%). On the other hand, theanti-TIGIT antibodies 4.1D3, 7.4A3.C96S, h1.6132.L1H1, and h1.10A5.L1H1were found to possess acceptable stability properties in the MA studies.

TABLE 8 MA stability summary for OMT-derived anti-TIGIT antibody clones4.1A4.C96S 4.1D3 7.4A3.C96S AAPH Stress M in CDR-H1 is stable W inCDR-H1 is stable M in CDR-H1 is stable W in CDR-H2 is stable W in CDR-H2is stable W in CDR-H2 is stable W in CDR-L2 is stable Thermal NY inCDR-H1 is stable DS in CDR-H1 is stable N(52)T in CDR-H2 is stableStress N(52)T in CDR-H2 are NS in CDR-H1 is stable N(54)T in CDR-H2 isstable pH 5.5 stable DY in CDR-H2 is stable NP in CDR-H2 is stable (2weeks) N(54)T in CDR-H2 are DY in CDR-H3 is stable DS in CDR-H3 isstable Peptide map stable NN in CDR-L1 is stable analysis DY in CDR-H3is stable NS in CDR-L3 is unstable - 15.7% increase in deamidation SizeMonomer loss (0.5%) is Monomer loss (0.6%) is Monomer loss (1.1%) isCharge acceptable acceptable acceptable LC/MS Main peak loss (16.7%) isMain peak loss (11.1%) Main peak loss (7.3%) is unacceptable isacceptable acceptable Masses are as expected Masses are as expectedMasses are as expected Thermal NY in CDR-H1 no change DS CDR-H1 2.8%N(52)T in CDR-H2 no Stress N(52)T & N(54)T in CDR- increase inisomerization change pH 7.4 H2 no change NS CDR-H1 no change N(54)T inCDR-H2 no (2 weeks) DY in CDR-H3 no change DY in CDR-H2 no changePeptide map NS in CDR-L3 9.4% change NP in CDR-H2 no change analysisincrease in deamidation DY in CDR-H3 2.7% DS in CDR-H3 no changeincrease in isomerization NN in CDR-L1 no change Size Monomer loss(2.7%) Monomer loss (0.9%) Monomer loss (2.3%) Charge Main peak loss(21.9%) Main peak loss (27.7%) Main peak loss (15.7%) LC/MS Masses areas expected Masses are as expected Masses are as expected

TABLE 9-1 MA stability summary for SD-derived anti-TIGIT antibody clonesh7E7.L1H1 h15C8.L1H1 AAPH Stress No oxidation hotspots No oxidationhotspots Thermal DP in CDR-H2 is stable DP in CDR-H2 is stable Stress NGin CDR-H2 is stable NG in CDR-H2 is stable pH 5.5 NT in CDR-L2 is stableDG in CDR-H3 is unstable (2 weeks) NL in CDR-L2 is stable (14.7%increase in Peptide map N(92)N(93)G in CDR-L3 isomerization (3.8% toanalysis is stable - high initial 18.5%) deamidation 31.5% N(92)N(93)Gin CDR-L3 is stable - high initial deamidation 39.2% Size Monomer loss(0.8%) is Monomer loss (0.9%) is Charge acceptable acceptable LC/MS Mainpeak loss (6.2%) is Main peak loss (10.3%) is acceptable acceptableMasses are as expected Masses are as expected Thermal DP in CDR-H2 nochange DP in CDR-H2 no change Stress NG in CDR-H2 1.9% NG in CDR-H2 3.3%pH 7.4 increase in deamidation increase in deamidation (2 weeks) NT inCDR-L2 no change DG in CDR-H3 7.0% Peptide map NL in CDR-L2 no changeincrease in isomerization analysis N(92)N(93)G in CDR-L3 - N(92)N(93)Gin CDR-L3 - 24.3% increase in 15.5% increase in deamidation deamidationSize Monomer loss (0.5%) Monomer loss (1.5%) Charge Main peak loss(16.7%) Main peak loss (25.2%) LC/MS Masses are as expected Masses areas expected

TABLE 9-2 MA stability summary for SD-derived anti-TIGIT antibody clonesh6B2.L1H1 h10A5.L1H1 AAPH Stress M in CDR-H1 is stable M in CDR-H1 isstable W(52) and W(53) in CDR- W(52) and W(53) in CDR- H2 are stable H2are stable W in CDR-H3 is stable W in CDR-H3 is stable Thermal NG inCDR-H2 is stable NG in CDR-H2 is stable Stress NT in CDR-H2 is stable NTin CDR-H2 is stable pH 5.5 (2 NP in CDR-H2 is stable NP in CDR-H2 isstable weeks) NS is CDR-L3 is stable NS is CDR-L3 is stable Peptide mapanalysis Size Monomer loss (0.2%) is Monomer loss (0.1%) is Chargeacceptable acceptable LC/MS Main peak loss (2.4%) is Main peak loss(6.3%) is acceptable acceptable Masses are as expected Masses are asexpected Thermal NG in CDR-H2 5.6% NG in CDR-H2 4.8% Stress increase indeamidation increase in deamidation pH 7.4 (2 NT in CDR-H2 no change NTin CDR-H2 no change weeks) NP in CDR-H2 no change NP in CDR-H2 no changePeptide map NS is CDR-L3 no change NS is CDR-L3 no change analysisMonomer loss (0.0%) Monomer loss (0.2%) Size Main peak loss (9.9%) Mainpeak loss (14.5%) Charge Masses are as expected Masses are as expectedLC/MS

Example 6. Structural and Functional TIGIT Epitope Mapping

To better understand how the 4.1D3 anti-TIGIT antibody and its optimizedvariants (e.g., 4.1D3.Q1E), in particular, are capable of exemplarycross-reactivity (e.g., cross-reactivity between human, cyno, and rabbitTIGIT), high affinity for TIGIT, and robust blocking of TIGIT-PVR andTIGIT-CD226 interaction/function while exhibiting pharmacokinetic andmolecular assessment properties, the crystal structure of 4.1D3 incomplex with TIGIT was determined and compared to the crystal structuresof mouse-derived anti-TIGIT antibody 1A5 bound to TIGIT and ahamster-derived anti-TIGIT antibody 10A7 bound to TIGIT, as described indetail below. Alanine scanning mutagenesis experiments of the TIGITinterface were also performed to identify functional epitopic residues.

A. TIGIT and Fab Expression, Purification, and Crystallization

Human TIGIT residues 23-128 was expressed and purified as described(see, e.g., Stengel et al. PNAS. 109(14): 5399-5404, 2012). Fabfragments including heavy and light chains were expressed and purifiedas described (see, e.g., Carter et al. Nat. Biotech. 1992). PurifiedTIGIT was mixed with an excess of purified Fab fragment to generate theTIGIT/Fab complex. The complex was then further purified on a sizeexclusion column equilibrated in HEPES buffered saline (10 mM HEPES, pH7.5 and 100 mM NaCl) to generate a sample containing only 1:1 complexesof TIGIT and Fab. Each of the TIGIT/Fab complexes were then concentratedfor crystallization and subjected to standard techniques of highthroughput vapor diffusion crystallization screening. For the TIGIT/10A7complex, the sample was concentrated to approximately 25 mg/mL and foundto crystallize in 0.1 M HEPES pH 7.5, 20% PEG 4000, and 10% isopropanol.For the TIGIT/1A5 complex, the sample was concentrated to approximately25 mg/mL and found to crystallize in 0.05 M HEPES pH 7.0, 12% PEG3350,and 1% Tryptone. For the TIGIT/4.1D3 complex, the sample wasconcentrated to approximately 25 mg/mL and found to crystallize in 0.1 MHEPES pH 7.5, 10% PEG 6000, and 5% MPD. Crystals were cryoprotected withglycerol and flash frozen in liquid nitrogen according to standardtechniques for data collection.

B. Data Collection and Structure Solution

X-ray diffraction data was collected under cryo cooled conditions at 100Kelvin using various synchotron X-ray radiation at the Advanced LightSource (Berkeley, Calif.) or Advanced Photon Source (Argonne, Ill.)according to standard methods. Diffraction images were processed andreduced using the data processing software XDS (Kabsch W. Acta Cryst. D.Biol. Crystl. 66: 125-132, 2010). Models were generated using themolecular replacement technique with the program PHASER. The structureof human TIGIT (Stengel et al PNAS 2012) and Fab antibody model(Nakamura et al Cell Host Microbe 2013) were used as search models. Thestructures underwent iterative rounds of model adjustment using theprogram COOT and refinement using the Phenix.refine or BUSTER programs.Models were refined to acceptable R and R free values and Ramachandranstatistics (calculated by Molprobity). Data processing and refinementstatistics can be found in Table 10.

TABLE 10 Data Collection and Refinement Statistics TIGIT/10A7 TIGIT/1A5TIGIT/1D3 Data collection ALS 5.0.2 APS LS-CAT 21ID-G APS 22ID-D Spacegroup P21 P1 14 Cell dimensions a, b, c (Å) 53.02, 76.99, 136.91 74.60,88.78, 101.10 212.39, 212.39, 66.52 α, β, y (°) 90, 90.6, 90 101.1,101.5, 110.5 90, 90, 90 Resolution (Å) 136.90-1.84 (1.94-1.84)*81.11-2.77 (2.92-2.77)* 106.19-1.91 (2.01-1.91)* R_(sym) or R_(merge)0.063 (0.512) 0.062 (0.558) 0.089 (0.973) I/σI 12.7 (2.4) 12.1 (2.3)12.8 (1.8) Completeness (%) 96.4 (95.7) 97.8 (97.1) 93.8 (69.3)Redundancy 3.8 (3.8) 2.2 (2.1) 7.4 (6.6) Refinement Resolution (Å)136.9-1.85 81.11-2.77 27.96-1.91 No. reflections 90418 58936 107910R_(work)/R_(free) 17.8/21.8% 19.9/27.1% 18.6/21.6% r.m.s. deviationsBond lengths (Å) 0.010 0.010 0.010 Bond angles (°) 1.16 1.26 1.13Ramachandran Most favorable 97.0% 91.2% 97.4% Disallowed 0.3% 1.4% 0.1%*Values in parentheses are for highest-resolution shell.

C. The Structure of 4.1D3 Bound to Human TIGIT

The 4.1D3 Fab in complex with TIGIT crystallized in the 14 space group,with two complexes in the asymmetric unit, and diffracted to 1.91 Å.Overlay of the two individual complexes in the asymmetric unit showsonly minor changes in main chain positioning between the individualcopies. The structure of 4.1D3 bound to human TIGIT (FIG. 6A) shows that4.1D3 sterically interferes with PVR binding (FIGS. 6B and 6C). Theburied surface area between 4.1D3 and TIGIT is approximately 1630 Å².4.1D3 light chain interactions are clustered around TIGIT residues77-82, with CDR L1 Tyr27d and CDR L3 Tyr92, Ser93, Thr94, and Phe96contacting TIGIT residues Ile77, Pro79, Ser80, and Lys82. CDR L2 doesnot make any contact with TIGIT. 4.1D3 heavy chain interactions areprimarily mediated by CDR H2 and H3 and make more extensive contact withTIGIT. 4.1D3 CDR H1 Asn32 contacts TIGIT Leu73. 4.1D3 CDR H2 Tyr52,Arg52b, Phe53, Lys54, Tyr56, and Asp58 contact TIGIT residues Thr55,Gln56, Asn58, Glu60, Asp63, Gln64, Leu65, Ile68, Asn70, Ser80, andHis111. 4.1D3 CDR H3 residues Tyr99, Asp100, Leu100a Leu100b, andAla100c contact TIGIT residues Leu65, Ala67, Ile68, Leu73, His76, Ile77,Ser78, Pro79, and Lys82. 4.1D3 interacts with TIGIT using a combinationof non-polar and polar interactions. Light chain residue CDR L3 Thr94forms polar contacts with TIGIT Pro79 and Ser80. Heavy chain CDR H2residues Arg52b and Tyr56 form hydrogen bonds with TIGIT residues Asn58and Glu60. Heavy chain CDR H3 residue Leu100a forms a hydrogen bond withTIGIT His76. Heavy chain CDR H2 residue Lys54 and CDR H3 residue Asp100form salt bridges with TIGIT residues Glu60 and Lys82, respectively. Seealso FIGS. 6D and 6E.

Based on the crystal structure of the 4.1D3/TIGIT complex, the residuesof TIGIT that are contacted by 4.1D3 (i.e., the epitopic residues ofTIGIT bound by 4.1D3) and the residues of 4.1D3 that are contacted byTIGIT (i.e., the paratopic residues of 4.1D3 contacted by TIGIT) weredetermined. Tables 11 and 12, below, show the residues of TIGIT and thelight or heavy chain residues of 4.1D3 to which they contact, asassessed using a contact distance stringency of 3.7 Å, a point at whichvan der Waals (non-polar) interaction forces are highest.

TABLE 11 Epitopic residues of TIGIT and their corresponding paratopicresidues on the light chain of 4.1D3 TIGIT 4.1D3 Light chain Ile 77 Tyr 27d Pro 79 Tyr 92 Ser 93 Thr 94 Ser 80 Thr 94 Lys 82 Tyr  27d

TABLE 12 Epitopic residues of TIGIT and their corresponding paratopicresidues on the heavy chain 5 of 4.1D3 TIGIT 4.1D3 Heavy chain Thr 55Phe 53 Gln 56 Phe 53 Asn 58 Arg  52b Tyr 56 Glu 60 Lys 54 Tyr 56 Leu 65Leu 100b Ile 68 Leu 100a Leu 73 Leu 100a His 76 Tyr 99 Asp 100  Leu 100aLeu 100b Ser 78 Leu 100b Pro 79 Leu 100b Ser 80 Asp 58 Lys 82 Asp 100 His 111 Phe 53

Alanine scanning of the human TIGIT interface was also performed, withalanine mutations of TIGIT residues made for Gln53, Gln56, Glu60, Leu65,Ile68, Asn70, Leu73, His76, His111, Tyr113, and Thr117. These mutants,along with wild-type, were tested for binding to the 4.1D3 Fab fragment.In this experiment, mutation of TIGIT residues Glu60, Leu65, and Ile68reduced 4.1D3 binding greater than 10 fold (FIGS. 7A, 7B, and 7E).Mutation of TIGIT residues Gln56, Asn70, Leu73, His111, and Tyr113reduced 4.1D3 binding between 1 and 10 fold (FIGS. 7A, 7B, and 7E).Mutation of TIGIT Gln53, His76, and Thr117 did not affect 4.1D3 binding(FIGS. 7A, 7B, and 7E). This analysis agreed with the crystal structureanalysis, with the TIGIT residues that most affected 4.1D3 binding foundto interact with 4.1D3 in the structure.

D. The Structure of 1A5 Bound to Human TIGIT

The 1A5 Fab complex with TIGIT crystallized in the P1 space group, withfour complexes in the asymmetric unit, and diffracted to 2.77 Å. Overlayof the four individual complexes in the asymmetric unit shows only minorchanges in main chain positioning between the individual copies. Thestructure of 1A5 bound to human TIGIT (FIG. 8) shows that 1A5 stericallyinterferes with PVR binding, but the epitope on human TIGIT to which 1A5binds is not identical to that of 4.1D3 (FIG. 9). The buried surfacearea between 1A5 and TIGIT is approximately 1715 Å². 1A5 light chaininteractions with TIGIT are primarily clustered between residues 109 and119, with one outlying contact with Glu60. 1A5 light chain CDR L1 has asingle contact residue, Trp32, that contacts TIGIT residues Ile109,Thr117, and Thr119. 1A5 CDR L2 residue Lys50 contacts TIGIT Glu60. 1A5CDR L3 residues Gly91, Gln92, Ser93, and Tyr94 contact TIGIT residuesThr112, Tyr113, Pro114, Asp115, Gly116, and Thr117. For the 10A7 heavychain, CDR H1 residues Thr30 and Asp31 make a contact with TIGIT residueLeu73. 10A7 CDR H2 residues Tyr52, Val53, Ser54, Tyr58, and Tyr59 makecontact with TIGIT residues Gln53, Thr55, Asp72, Leu73, and Tyr113. 10A7CDR H3 residues Phe97, Arg98, Pro100, and Trp100a make contact withTIGIT residues Gln56, Asn58, Glu60, Asp63, Gln64, Leu65, Ile68, Leu73,His76, and His111. Contacts between 1A5 and TIGIT are primarilynon-polar in nature, with the two exceptions being a hydrogen bondbetween 1A5 CDR H2 residue Ser54 and TIGIT residue Asp72, and a saltbridge between 1A5 CDR H3 residue Arg98 and TIGIT residue Glu60.

Based on the crystal structure of the 1A5/TIGIT complex, the residues ofTIGIT that are contacted by 1A5 (i.e., the epitopic residues of TIGITbound by 1A5) and the residues of 1A5 that are contacted by TIGIT (i.e.,the paratopic residues of 1A5 contacted by TIGIT) were determined.Tables 13 and 14, below, show the residues of TIGIT and the light orheavy chain residues of 1A5 to which they contact, as assessed using acontact distance stringency of 3.7 Å, a point at which van der Waals(non-polar) interaction forces are highest.

TABLE 13 Epitopic residues of TIGIT and their corresponding paratopicresidues on the light chain of 1A5 TIGIT 1A5 Light chain Thr 112 Tyr 94Tyr 113 Tyr 94 Pro 114 Tyr 94 Gly 116 Gln 92 Thr 117 Trp 32 Gly 91 Gln92

TABLE 14 Epitopic residues of TIGIT and their corresponding paratopicresidues on the heavy chain of 1A5 TIGIT 1A5 Heavy chain Gln 53 Tyr 58Thr 55 Tyr 52 Tyr 58 Gln 56 Phe 97 Arg 98 Pro 100  Asn 58 Arg 98 Glu 60Arg 98 Leu 65 Arg 98 Asp 72 Tyr 52 Val 53 Ser 54 Leu 73 Thr 30 Asp 31Tyr 113  Tyr 58 Tyr 59

Alanine scanning of the TIGIT interface was also performed, with alaninemutations of TIGIT residues made for Gln53, Gln56, Glu60, Leu65, Ile68,Asn70, Leu73, His76, His111, Tyr113, and Thr117. These mutants, alongwith wild-type, were tested for binding to the 1A5 Fab fragment. In thisexperiment, mutation of TIGIT residues Gln56, Glu60, Leu65, Ile68, andTyr113 reduced 1A5 binding greater than 10 fold (FIGS. 7A, 7B, and 7D).Mutation of TIGIT residues Leu73, His76, Asn70, His111, and Thr117reduced 1A5 binding between 1 and 10 fold (FIGS. 7A, 7B, and 7D). Onlymutation of TIGIT Gln53 did not affect 1A5 binding (FIGS. 7A, 7B, and7D). This analysis agreed with the crystal structure analysis, with manyof the TIGIT residues that most affected 1A5 binding found to interactwith 1A5 in the structure.

E. The Structure of 10A7 Bound to Human TIGIT

The 10A7 Fab/TIGIT complex crystallized in the P21 space group, with two10A7/TIGIT complexes in the asymmetric unit, and diffracted to 1.85 Å.Overlay of the two complexes shows only minor differences in main chainpositioning for both TIGIT and 10A7. The structure of 10A7 bound tohuman TIGIT (FIG. 10A) shows that 10A7 sterically interferes with PVRbinding (FIG. 10B), but the epitope on human TIGIT to which 10A7 bindsis not identical to that of 4.1D3 or 1A5 (FIG. 9). The buried surfacearea between 10A7 and TIGIT is approximately 1420 Å². TIGIT contactresidues cluster between Leu65 and Ile77, with two outlying 10A7 CDRcontacts with TIGIT Gln56 and Pro87. The light chain CDR L1 has as sixamino acid insertion and utilizes residues Tyr27d, Gly27f, Val28, Lys29,and Leu32 to make contacts with TIGIT residues Gln56, Leu65, Ile68,Asn70, Leu73, His76, Ser78, Ile77, and Pro79. CDR L2 only makes twocontacts via Tyr50 and Ile53 with TIGIT His76, Ile77, and Pro79. CDR L3Gly91, Ile92, Asn93, and Asn94 contact TIGIT residues Asp72, Leu73, andHis76. For the heavy chain CDRs, no H1 residues contact TIGIT. For CDRH2, Phe50 and Arg52 contact Asn70, Ala71, Asp72, Leu73, and Gly74. ForCDR H3, residues Arg95, Leu97, Gly98, His99, and Asn100 contact TIGITresidues Leu73, Gly74, Trp75, His76, Ile77, and Pro87. Overall theinteractions between the 10A7 light chain and TIGIT were primarilyhydrophobic in nature, with one hydrogen bond contact between CDR L2Tyr50 hydroxyl group and the backbone carbonyl of Ile77. For the 10A7heavy chain, polar contacts are found between Arg52 and the backbonecarbonyl groups of TIGIT Ala71 and Asp72, as well as between Arg95 andthe backbone carbonyl groups of Gly74 and Trp75.

Based on the crystal structure of the 10A7/TIGIT complex, the residuesof TIGIT that are contacted by 10A7 (i.e., the epitopic residues ofTIGIT bound by 10A7) and the residues of 10A7 that are contacted byTIGIT (i.e., the paratopic residues of 10A7 contacted by TIGIT) weredetermined. Tables 15 and 16, below, show the residues of TIGIT and thelight or heavy chain residues of 10A7 to which they contact, as assessedusing a contact distance stringency of 3.7 Å, a point at which van derWaals (non-polar) interaction forces are highest.

TABLE 15 Epitopic residues of TIGIT and their corresponding paratopicresidues on the light chain of 10A7 TIGIT 10A7 Light chain Asp 72 Asn 94Leu 73 Gly 91 Ile 92 His 76 Tyr  27d Leu 32 Tyr 50 Ile 77 Tyr 50 Pro 79Glu 30 Tyr 50 Ile 53

TABLE 16 Epitopic residues of TIGIT and their corresponding paratopicresidues on the heavy chain of 10A7 TIGIT 10A7 Heavy chain Ala 71 Arg 52Phe 58 Asp 72 Arg 52 Leu 73 Arg 52 Arg 95 Asn 100  Gly 74 Arg 52 Trp 75Arg 95 Leu 97 Gly 98 His 99 His 76 Gly 98 Asn 100  Ile 77 Gly 98 Pro 87Leu 97

Alanine scanning of the TIGIT interface was also performed, with alaninemutations of TIGIT residues made for Gln53, Gln56, Glu60, Leu65, Ile68,Asn70, Leu73, His76, His111, Tyr113, and Thr117. These mutants, alongwith wild-type, were tested for binding to the 10A7 Fab fragment. Inthis experiment, only Leu73Ala and His76Ala mutations affected 10A7binding greater than 10 fold (FIGS. 7A-7C). Mutation of the otherresidues listed did not significantly affect 10A7 binding (FIGS. 7A-7C).This agreed closely with the crystal structure of 10A7 bound to TIGIT,in which Leu73 and His76 were found directly in the epitope bound by10A7.

F. The Anti-TIGIT Antibodies 4.1D3, 1A5, and 10A7 Recognize TIGIT atUnique Epitopes

The structural studies described above demonstrate that the threeanti-TIGIT antibodies 4.1D3, 1A5, and 10A7, recognize TIGIT at uniqueepitopes, which may explain their distinct functional properties andcharacteristics. As shown below in Table 17, for example, 4.1D3 binds toTIGIT at residues Ser78, Ser80, and Lys82, which are not bound by eitherthe 1A5 or the 10A7 antibodies. FIG. 11 is a structural rendering,showing that residues Ser78, Ser80, and Lys82 of TIGIT are intimatelycontacted by 4.1D3, but not by 1A5 or 10A7.

TABLE 17 Epitopic residues of TIGIT within 3.7Å for the 4.1D3, 1A5, and10A7 anti-TIGIT antibodies 4.1D3 1A5 10A7 53 55 55 56 56 58 58 60 60 6565 68 71 72 72 73 73 73 74 75 76 76 77 77 78 79 79 80 82 87 111  112 113  114  116  117 

Example 7. Characterization of TIGIT, PD-1, and CD226 Expression onImmune Cells

Next, we characterized the expression of TIGIT, PD-1, and CD226 on CD4+and CD8+ T cells from bone marrow specimens of multiple myeloma (MM)pateints using multi-color flow cytometry. Cells were isolated fromfrozen bone marrow procured from MM patients (n=10). Two of the MMpatients tested had samples collected at diagnosis and again uponremission. Frozen bone marrow from healthy donors was used as a control(n=8). In these experiments, the bone marrow samples were stained withthe following fluorescently conjugated monoclonal antibodies: BV605(PD-1), Alexa Fluor 488 (FoxP3), PE-DNAM-1 (CD226), PE-Cy7 (CD45),BUV737 (CD8), PerCP-eF710 (CD4), APC (TIGIT), Brilliant Violet 421(NKp46), Brilliant Violet 421 (CD56), Brilliant Violet 510 (CD3),Brilliant Violet 510 (CD38), PE-Cy7 (CD319), PE (CD19), and LIVE/DEADFixableNear-IR Dead Cell Stain (Life Technologies, ThermoFisher).

TIGIT, PD-1, and CD226 were shown to be highly expressed on CD4+ andCD8+ T cells obtained from the bone marrow of MM patients compared toCD4+ and CD8+ T cells obtained from the peripheral blood of healthypatients (FIG. 12). CD4+ and CD8+ T cells obtained from the bone marrowof MM patients were also determined to co-express both TIGIT and PD-1(FIG. 13). Given this expression pattern, the anti-TIGIT antibodies ofthe invention are useful for treating patients having an immune-relateddisease or cancer (e.g., a myeloma, e.g., MM), either as a monotherapyor in combination with an additional therapeutic agent, such as a PD-1axis binding antagonist (e.g., a PD-L1 binding antagonist, e.g., ananti-PD-L1 antibody, e.g., MPDL3280A (atezolizumab)).

OTHER EMBODIMENTS

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. It is understood that various otherembodiments may be practiced, given the general description providedabove. The disclosures of all patent and scientific literature citedherein are expressly incorporated in their entirety by reference.

What is claimed is:
 1. An antibody that specifically binds to humanTIGIT, wherein the antibody binds to an epitope on human TIGITcomprising one or more of amino acid residues Ser78, Ser80, and Lys82 ofhuman TIGIT.
 2. The antibody of claim 1, wherein the epitope comprisesamino acid residues Ser80 and Lys82 of human TIGIT.
 3. The antibody ofclaim 1 or 2, wherein the epitope comprises amino acid residues Ser78,Ser80, and Lys82 of human TIGIT.
 4. The antibody of any one of claims1-3, wherein the epitope further comprises amino acid residue Ala67 ofhuman TIGIT.
 5. The antibody of any one of claims 1-4, wherein theepitope further comprises one or more additional amino acid residuesselected from the group consisting of Glu60, Leu65, and Ile68 of humanTIGIT.
 6. The antibody of any one of claims 1-5, wherein the epitopefurther comprises one or more additional amino acid residues selectedfrom the group consisting of Gln56, Asn70, Leu73, and His111 of humanTIGIT.
 7. The antibody of any one of claims 1-6, wherein the epitopefurther comprises one or more additional amino acid residues selectedfrom the group consisting of Thr55, Asn58, Asp63, Gln64, His76, Ile77,and Pro79 of human TIGIT.
 8. The antibody of any one of claims 1-7,wherein the epitope consists of amino acid residues Thr55, Gln56, Asn58,Glu60, Asp63, Gln64, Leu65, Ala67, Ile68, Asn70, Leu73, His76, Ile77,Ser78, Pro79, Ser80, Lys82, and His111 of human TIGIT.
 9. An antibodythat specifically binds to human TIGIT, wherein the antibody binds to anepitope on human TIGIT comprising one or more of amino acid residuesThr55, Ser80, and Lys82 of human TIGIT.
 10. The antibody of claim 9,wherein the epitope comprises amino acid residue Lys82 of human TIGIT.11. The antibody of claim 9 or 10, wherein the epitope comprises aminoacid residues Thr55, Ser80, and Lys82 of human TIGIT.
 12. The antibodyof any one of claims 9-11, wherein the epitope further comprises aminoacid residue Gln56 of human TIGIT.
 13. The antibody of any one of claims9-12, wherein the epitope further comprises amino acid residue Ile77 orPro79 of human TIGIT.
 14. The antibody of claim 13, wherein the epitopefurther comprises amino acid residues Ile77 and Pro79 of human TIGIT.15. The antibody of any one of claims 9-14, wherein the epitope furthercomprises amino acid residue Asn58 or Glu60 of human TIGIT.
 16. Theantibody of claim 15, wherein the epitope further comprises amino acidresidues Asn58 and Glu60 of human TIGIT.
 17. The antibody of any one ofclaims 9-16, wherein the epitope further comprises one or moreadditional amino acid residues selected from the group consisting ofLeu65, Ile68, Leu73, His76, Ser78, and His111 of human TIGIT.
 18. Theantibody of claim 17, wherein the epitope further comprises amino acidresidues Leu65, Ile68, Leu73, His76, Ser78, and His111 of human TIGIT.19. The antibody of any one of claims 9-18, wherein the epitope consistsof Thr55, Gln56, Asn58, Glu60, Leu65, Ile68, Leu73, His76, Ile77, Ser78,Pro79, Ser80, Lys82, and His111 of human TIGIT.
 20. An antibody thatspecifically binds to human TIGIT, wherein the antibody comprises aparatope comprising one or more amino acid residues selected from thegroup consisting of heavy chain variable region amino acid residuesAsn32, Tyr52, Arg52b, Phe53, Lys54, Tyr56, Asp58, Tyr99, Asp100,Leu100a, Leu100b, and Ala100c and light chain variable region amino acidresidues Tyr27d, Tyr92, Ser93, Thr94, and Phe96.
 21. The antibody ofclaim 20, wherein the paratope consists of heavy chain variable regionamino acid residues Asn32, Tyr52, Arg52b, Phe53, Lys54, Tyr56, Asp58,Tyr99, Asp100, Leu100a, Leu100b, and Ala100c and light chain variableregion amino acid residues Tyr27d, Tyr92, Ser93, Thr94, and Phe96. 22.An antibody that specifically binds to human TIGIT, wherein the antibodybinds to an epitope on human TIGIT comprising one or more amino acidresidues selected from the group consisting of Gln53, His111, and Tyr113of human TIGIT.
 23. The antibody of claim 22, wherein the epitopefurther comprises Gln56 of human TIGIT.
 24. The antibody of claim 22 or23, wherein the epitope further comprises Glu60, Leu65, Ile68, Asn70,Leu73, and His76 of human TIGIT.
 25. An antibody that specifically bindsto human TIGIT, wherein the antibody comprises the following sixhypervariable regions (HVRs): an HVR-H1 comprising the amino acidsequence of SNSAAWN (SEQ ID NO: 1); an HVR-H2 comprising the amino acidsequence of KTYYRFKWYSDYAVSVKG (SEQ ID NO: 2); an HVR-H3 comprising theamino acid sequence of ESTTYDLLAGPFDY (SEQ ID NO: 3); an HVR-L1comprising the amino acid sequence of KSSQTVLYSSNNKKYLA (SEQ ID NO: 4);an HVR-L2 comprising the amino acid sequence of WASTRES (SEQ ID NO: 5);and an HVR-L3 comprising the amino acid sequence of QQYYSTPFT (SEQ IDNO: 6).
 26. The antibody of claim 25, wherein the antibody furthercomprises the following light chain variable region framework regions(FRs): an FR-L1 comprising the amino acid sequence ofDIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 7); an FR-L2 comprising the aminoacid sequence of WYQQKPGQPPNLLIY (SEQ ID NO: 8); an FR-L3 comprising theamino acid sequence of GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 9);and an FR-L4 comprising the amino acid sequence of FGPGTKVEIK (SEQ IDNO: 10).
 27. The antibody of claim 25 or 26, wherein the antibodyfurther comprises the following heavy chain variable region FRs: anFR-H1 comprising the amino acid sequence ofX₁VQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 11), wherein X₁ is Q or E;an FR-H2 comprising the amino acid sequence of WIRQSPSRGLEWLG (SEQ IDNO: 12); an FR-H3 comprising the amino acid sequence ofRITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 13); and an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 14). 28.The antibody of claim 27, wherein the antibody further comprises thefollowing heavy chain variable region FRs: an FR-H1 comprising the aminoacid sequence of EVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 15); anFR-H2 comprising the amino acid sequence of WIRQSPSRGLEWLG (SEQ ID NO:12); an FR-H3 comprising the amino acid sequence ofRITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 13); and an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 14). 29.The antibody of claim 27, wherein the antibody further comprises thefollowing heavy chain variable region FRs: an FR-H1 comprising the aminoacid sequence of QVQLQQSGPGLVKPSQTLSLTCAISGDSVS (SEQ ID NO: 16); anFR-H2 comprising the amino acid sequence of WIRQSPSRGLEWLG (SEQ ID NO:12); an FR-H3 comprising the amino acid sequence ofRITINPDTSKNQFSLQLNSVTPEDTAVFYCTR (SEQ ID NO: 13); and an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 14). 30.An antibody that specifically binds to human TIGIT, wherein the antibodycomprises the following six HVRs: an HVR-H1 comprising the amino acidsequence of SYPMN (SEQ ID NO: 17); an HVR-H2 comprising the amino acidsequence of WINTNTGNPTYVQGFTG (SEQ ID NO: 18); an HVR-H3 comprising theamino acid sequence of TGGHTYDSYAFDV (SEQ ID NO: 19); an HVR-L1comprising the amino acid sequence of RASQVISSSLA (SEQ ID NO: 20); anHVR-L2 comprising the amino acid sequence of AASTLQS (SEQ ID NO: 21);and an HVR-L3 comprising the amino acid sequence of QHLHGYPX₁N (SEQ IDNO: 22), wherein X₁ is C or S.
 31. The antibody of claim 30, whereinantibody comprises the following six HVRs: an HVR-H1 comprising theamino acid sequence of SYPMN (SEQ ID NO: 17); an HVR-H2 comprising theamino acid sequence of WINTNTGNPTYVQGFTG (SEQ ID NO: 18); an HVR-H3comprising the amino acid sequence of TGGHTYDSYAFDV (SEQ ID NO: 19); anHVR-L1 comprising the amino acid sequence of RASQVISSSLA (SEQ ID NO:20); an HVR-L2 comprising the amino acid sequence of AASTLQS (SEQ ID NO:21); and an HVR-L3 comprising the amino acid sequence of QHLHGYPSN (SEQID NO: 23).
 32. The antibody of any one of claim 30 or 31, wherein theantibody further comprises the following heavy chain variable regionFRs: an FR-H1 comprising the amino acid sequence ofEVQLVQSGSDLKKPGASVRVSCKASGYTFT (SEQ ID NO: 24); an FR-H2 comprising theamino acid sequence of WVRQAPGHGLEWMG (SEQ ID NO: 25); an FR-H3comprising the amino acid sequence of RFVFSLDTSVNTAYLQISSLKAEDTAVYFCAR(SEQ ID NO: 26); and an FR-H4 comprising the amino acid sequence ofWGQGTMVTVSS (SEQ ID NO: 27).
 33. The antibody of claim 30, whereinantibody comprises the following six HVRs: an HVR-H1 comprising theamino acid sequence of SYPMN (SEQ ID NO: 17); an HVR-H2 comprising theamino acid sequence of WINTNTGNPTYVQGFTG (SEQ ID NO: 18); an HVR-H3comprising the amino acid sequence of TGGHTYDSYAFDV (SEQ ID NO: 19); anHVR-L1 comprising the amino acid sequence of RASQVISSSLA (SEQ ID NO:20); an HVR-L2 comprising the amino acid sequence of AASTLQS (SEQ ID NO:21); and an HVR-L3 comprising the amino acid sequence of QHLHGYPCN (SEQID NO: 28).
 34. The antibody of claim 33, wherein the antibody furthercomprises the following heavy chain variable region FRs: an FR-H1comprising the amino acid sequence of QVQLVQSGSDLKKPGASVRVSCKASGYTFT(SEQ ID NO: 29); an FR-H2 comprising the amino acid sequence ofWVRQAPGHGLEWMG (SEQ ID NO: 25); an FR-H3 comprising the amino acidsequence of RFVFSLDTSVNTAYLQISSLKAEDTAVYFCAR (SEQ ID NO: 26); and anFR-H4 comprising the amino acid sequence of WGQGTMVTVSS (SEQ ID NO: 27).35. The antibody of any one of claims 30-34, wherein the antibodyfurther comprises the following light chain variable region FRs: anFR-L1 comprising the amino acid sequence of DIQLTQSPTFLSASVGDRVTITC (SEQID NO: 30); an FR-L2 comprising the amino acid sequence ofWYQQNPGKAPKLLIY (SEQ ID NO: 31); an FR-L3 comprising the amino acidsequence of GVPSRFSGSGSGTEFTLTISSLQPEDFVTYYC (SEQ ID NO: 32); and anFR-L4 comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 33).36. An antibody that specifically binds to human TIGIT, wherein theantibody comprises (a) a heavy chain variable region (VH) having atleast 95% sequence identity to the amino acid sequence of SEQ ID NO: 34or 35; (b) a light chain variable region (VL) having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 36; or (c) aheavy chain variable region as in (a) and a light chain variable regionas in (b).
 37. The antibody of claim 36, wherein the antibody comprises(a) a heavy chain variable region (VH) having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO: 34; (b) a light chainvariable region (VL) having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 36; or (c) a heavy chain variable region asin (a) and a light chain variable region as in (b).
 38. The antibody ofclaim 36, wherein the antibody comprises (a) a heavy chain variableregion (VH) having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 35; (b) a light chain variable region (VL) havingat least 95% sequence identity to the amino acid sequence of SEQ ID NO:36; or (c) a heavy chain variable region as in (a) and a light chainvariable region as in (b).
 39. An antibody that specifically binds tohuman TIGIT, wherein the antibody comprises (a) a heavy chain variableregion (VH) having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 37; (b) a light chain variable region (VL) havingat least 95% sequence identity to the amino acid sequence of SEQ ID NO:38; or (c) a heavy chain variable region as in (a) and a light chainvariable region as in (b).
 40. An antibody that specifically binds tohuman TIGIT, wherein the antibody comprises (a) a heavy chain variableregion (VH) having at least 95% sequence identity to the amino acidsequence of SEQ ID NO: 39; (b) a light chain variable region (VL) havingat least 95% sequence identity to the amino acid sequence of SEQ ID NO:40; or (c) a heavy chain variable region as in (a) and a light chainvariable region as in (b).
 41. The antibody of any one of claims 1-21,25-29, and 36-38, wherein the antibody is capable of binding to rabbitTIGIT.
 42. The antibody of any one of claims 1-41, wherein the antibodyis capable of binding to both human TIGIT and cynomolgus monkey (cyno)TIGIT, but not murine TIGIT.
 43. The antibody of claim 42, wherein theantibody binds human TIGIT with a Kd of about 10 nM or lower and cynoTIGIT with a Kd of about 10 nM or lower.
 44. The antibody of claim 43,wherein the antibody binds human TIGIT with a Kd of about 0.1 nM toabout 1 nM and cyno TIGIT with a Kd of about 0.5 nM to about 1 nM. 45.The antibody of claim 44, wherein the antibody binds human TIGIT with aKd of about 0.1 nM or lower and cyno TIGIT with a Kd of about 0.5 nM orlower.
 46. The antibody of any one of claims 1-45, wherein the antibodyis an antagonist antibody.
 47. The antibody of claim 46, wherein theantagonist antibody specifically binds TIGIT and inhibits or blocksTIGIT interaction with poliovirus receptor (PVR).
 48. The antibody ofclaim 47, wherein the antagonist antibody inhibits intracellularsignaling mediated by TIGIT binding to PVR.
 49. The antibody of any oneof claims 46-48, wherein the antagonist antibody inhibits or blocksbinding of human TIGIT to human PVR with an IC50 value of 10 nM orlower.
 50. The antibody of any one of claims 46-49, wherein theantagonist antibody inhibits or blocks binding of human TIGIT to humanPVR with an IC50 value of 1 nM to about 10 nM.
 51. The antibody of anyone of claims 46-50, wherein the antagonist antibody inhibits or blocksbinding of cyno TIGIT to cyno PVR with an IC50 value of 50 nM or lower.52. The antibody of claim 51, wherein the antagonist antibody inhibitsor blocks binding of cyno TIGIT to cyno PVR with an IC50 value of 1 nMto about 50 nM.
 53. The antibody of claim 52, wherein the antagonistantibody inhibits or blocks binding of cyno TIGIT to cyno PVR with anIC50 value of 1 nM to about 5 nM.
 54. The antibody of any one of claims1-45, wherein the antibody is an agonist antibody.
 55. The antibody ofclaim 54, wherein the agonist antibody specifically binds TIGIT andstimulates the interaction of PVR with CD226 or CD96.
 56. The antibodyof claim 55, wherein the agonist antibody specifically binds TIGIT andstimulates the interaction of PVR with CD226 and CD96.
 57. The antibodyof claim 56, wherein the agonist antibody specifically binds TIGIT andstimulates the interaction of human PVR with human CD226 and human CD96.58. The antibody of claim 56, wherein the agonist antibody specificallybinds TIGIT and stimulates the interaction of cyno PVR with cyno CD226and cyno CD96.
 59. An isolated antibody that competes for binding toTIGIT with the antibody of any one of claims 1-58.
 60. An isolatedantibody that binds to the same epitope as the antibody of any one ofclaims 1-58.
 61. The antibody of any one of claims 1-60, wherein theantibody is monoclonal.
 62. The antibody of any one of claims 1-60,wherein the antibody is human, humanized, or chimeric.
 63. The antibodyof claim 62, wherein at least a portion of the framework sequence is ahuman consensus framework sequence.
 64. The antibody of any one ofclaims 1-63, wherein the antibody is a full-length antibody.
 65. Theantibody of any one of claims 1-64, wherein the antibody has a clearancefollowing intravenous injection of about 3 ml/kg/day to about 10ml/kg/day.
 66. The antibody of any one of claim 65, wherein the antibodyhas a clearance following intravenous injection of about 3 ml/kg/day toabout 8 ml/kg/day.
 67. The antibody of any one of claims 1-63, whereinthe antibody is an antibody fragment that binds TIGIT.
 68. The antibodyof claim 67, wherein the antibody fragment is selected from the groupconsisting of Fab, Fab′, Fab′-SH, Fv, single chain variable fragment(scFv), and (Fab′)₂ fragments.
 69. The antibody of any one of claims1-68, wherein the antibody is an IgG class antibody.
 70. The antibody ofclaim 69, wherein the IgG class antibody is an IgG1 subclass antibody.71. A polynucleotide encoding the antibody of any one of claims 1-70.72. A vector comprising the polynucleotide of claim
 71. 73. A host cellcomprising the vector of claim
 72. 74. The host cell of claim 73,wherein the host cell is prokaryotic.
 75. The host cell of claim 74,wherein the host cell is Escherichia coli.
 76. The host cell of claim73, wherein the host cell is eukaryotic.
 77. The host cell of claim 76,wherein the host cell is a 293 cell, a CHO cell, a yeast cell, or aplant cell.
 78. A method of producing the antibody of any one of claims1-70, the method comprising culturing the host cell of claim 73 in aculture medium.
 79. The method of claim 78, wherein the method furthercomprises recovering the antibody from the host cell or culture medium.80. An immunoconjugate comprising the antibody of any one of claims 1-70and a cytotoxic agent.
 81. A composition comprising the antibody of anyone of claims 1-70.
 82. The composition of claim 81, further comprisinga pharmaceutically acceptable carrier, excipient, or diluent.
 83. Thecomposition of claim 82, wherein the composition is a pharmaceuticalcomposition.
 84. The composition of any one of claims 81-83, wherein thecomposition further comprises a PD-1 axis binding antagonist or anadditional therapeutic agent.
 85. The antibody of any one of claims 1-70for use as a medicament.
 86. The antibody of any one of claims 1-70 foruse in treating or delaying progression of a cancer in a subject in needthereof.
 87. The antibody of claim 86, wherein the cancer is selectedfrom the group consisting of a non-small cell lung cancer, a small celllung cancer, a renal cell cancer, a colorectal cancer, an ovariancancer, a breast cancer, a pancreatic cancer, a gastric carcinoma, abladder cancer, an esophageal cancer, a mesothelioma, a melanoma, a headand neck cancer, a thyroid cancer, a sarcoma, a prostate cancer, aglioblastoma, a cervical cancer, a thymic carcinoma, a leukemia, alymphoma, a myeloma, mycoses fungoides, a merkel cell cancer, and ahematologic malignancy.
 88. The antibody of claim 87, wherein themyeloma is multiple myeloma (MM).
 89. The antibody of any one of claims1-70 for use in treating or delaying progression of an immune-relateddisease in a subject in need thereof.
 90. The antibody of claim 89,wherein the immune-related disease is associated with a T celldysfunctional disorder.
 91. The antibody of claim 90, wherein the T celldysfunctional disorder is characterized by T cell exhaustion.
 92. Theantibody of any one of claims 89-91, wherein the immune-related diseaseis selected from the group consisting of unresolved acute infection,chronic infection, and tumor immunity.
 93. The antibody of any one ofclaims 1-70 for use in increasing, enhancing, or stimulating an immuneresponse or function in a subject in need thereof.
 94. Use of theantibody of any one of claims 1-70 in the manufacture of a medicamentfor treating or delaying progression of a cancer in a subject in needthereof.
 95. The use of claim 94, wherein the cancer is selected fromthe group consisting of a non-small cell lung cancer, a small cell lungcancer, a renal cell cancer, a colorectal cancer, an ovarian cancer, abreast cancer, a pancreatic cancer, a gastric carcinoma, a bladdercancer, an esophageal cancer, a mesothelioma, a melanoma, a head andneck cancer, a thyroid cancer, a sarcoma, a prostate cancer, aglioblastoma, a cervical cancer, a thymic carcinoma, a leukemia, alymphoma, a myeloma, mycoses fungoides, a merkel cell cancer, and ahematologic malignancy.
 96. The use of claim 95, wherein the myeloma isMM.
 97. Use of the antibody of any one of claims 1-70 in the manufactureof a medicament for treating or delaying progression of animmune-related disease in a subject in need thereof.
 98. The use ofclaim 97, wherein the immune-related disease is associated with a T celldysfunctional disorder.
 99. The use of claim 98, wherein the T celldysfunctional disorder is characterized by T cell exhaustion.
 100. Theuse of any one of claims 97-99, wherein the immune-related disease isselected from the group consisting of unresolved acute infection,chronic infection, and tumor immunity.
 101. Use of the antibody of anyone of claims 1-70 in the manufacture of a medicament for increasing,enhancing, or stimulating an immune response or function in a subject inneed thereof.
 102. A method for treating or delaying progression of acancer in a subject, the method comprising administering to the subjectan effective amount of the antibody of any one of claims 1-70, therebytreating or delaying the progression of the cancer in the subject. 103.The method of claim 102, wherein the cancer is selected from the groupconsisting of a non-small cell lung cancer, a small cell lung cancer, arenal cell cancer, a colorectal cancer, an ovarian cancer, a breastcancer, a pancreatic cancer, a gastric carcinoma, a bladder cancer, anesophageal cancer, a mesothelioma, a melanoma, a head and neck cancer, athyroid cancer, a sarcoma, a prostate cancer, a glioblastoma, a cervicalcancer, a thymic carcinoma, a leukemia, a lymphoma, a myeloma, mycosesfungoides, a merkel cell cancer, and a hematologic malignancy.
 104. Themethod of claim 103, wherein the myeloma is MM.
 105. A method fortreating or delaying progression of an immune-related disease in asubject, the method comprising administering to the subject an effectiveamount of the antibody of any one of claims 1-70, thereby treating ordelaying the progression of the immune-related disease in the subject.106. The method of claim 105, wherein the immune-related disease isassociated with a T cell dysfunctional disorder.
 107. The method ofclaim 106, wherein the T cell dysfunctional disorder is characterized byT cell exhaustion.
 108. The method of any one of claims 105-107, whereinthe immune-related disease is selected from the group consisting ofunresolved acute infection, chronic infection, and tumor immunity. 109.A method of increasing, enhancing, or stimulating an immune response orfunction in a subject, the comprising administering to the subject aneffective amount of the antibody of any one of claims 1-70, therebyincreasing, enhancing, or stimulating an immune response or function inthe subject.
 110. The method of any one of 102-109, further comprisingadministering to the subject a PD-1 axis binding antagonist.
 111. Themethod of claim 110, wherein the PD-1 axis binding antagonist isadministered prior to or subsequent to the administration of theantibody.
 112. The method of claim 110, wherein the PD-1 axis bindingantagonist is administered concurrently with the antibody.
 113. Themethod of any one of claims 110-112, wherein the PD-1 axis bindingantagonist is selected from the group consisting of a PD-1 bindingantagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist.114. The method of claim 113, wherein the PD-1 axis binding antagonistis a PD-1 binding antagonist.
 115. The method of claim 114, wherein thePD-1 binding antagonist inhibits the binding of PD-1 to its ligandbinding partners.
 116. The method of claim 115, wherein the PD-1 bindingantagonist inhibits the binding of PD-1 to PD-L1.
 117. The method ofclaim 115, wherein the PD-1 binding antagonist inhibits the binding ofPD-1 to PD-L2.
 118. The method of claim 115, wherein the PD-1 bindingantagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. 119.The method of any one of claims 115-118, wherein the PD-1 bindingantagonist is an anti-PD-1 antibody.
 120. The method of claim 115,wherein the PD-1 binding antagonist is selected from the groupconsisting of MDX 1106 (nivolumab), MK-3475 (pembrolizumab), CT-011(pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108. 121.The method of claim 113, wherein the PD-1 axis binding antagonist is aPD-L1 binding antagonist.
 122. The method of claim 121, wherein thePD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1.
 123. Themethod of claim 121, wherein the PD-L1 binding antagonist inhibits thebinding of PD-L1 to B7-1.
 124. The method of claim 121, wherein thePD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 andB7-1.
 125. The method of claim 121-124, wherein the PD-L1 bindingantagonist is an anti-PD-L1 antibody.
 126. The method of claim 125,wherein the anti-PD-L1 antibody is selected from the group consistingof: MPDL3280A (atezolizumab), YW243.55.S70, MDX-1105, MEDI4736(durvalumab), and MSB0010718C (avelumab).
 127. The method of claim 126,wherein the antibody is MPDL3280A.
 128. The method of claim 113, whereinthe PD-1 axis binding antagonist is a PD-L2 binding antagonist.
 129. Themethod of claim 128, wherein the PD-L2 binding antagonist is ananti-PD-L2 antibody.
 130. The method of claim 128, wherein the PD-L2binding antagonist is an immunoadhesin.
 131. The method of any one ofclaims 102-130, further comprising administering to the subject an OX40binding agonist.
 132. The method of claim 131, wherein the OX40 bindingagonist is administered prior to or subsequent to the administration ofthe antibody and/or the PD-1 axis binding antagonist.
 133. The method ofclaim 131, wherein the OX40 binding agonist is administered concurrentlywith the antibody and/or the PD-1 axis binding antagonist.
 134. Themethod of any one of claims 131-133, wherein the OX40 binding agonist isselected from the group consisting of an OX40 agonist antibody, an OX40Lagonist fragment, an OX40 oligomeric receptor, and an OX40immunoadhesin.
 135. The method of claim 134, wherein the OX40 agonistantibody depletes cells that express human OX40.
 136. The method ofclaim 135, wherein the cells that express human OX40 are CD4+ effector Tcells.
 137. The method of claim 135, wherein the cells that expresshuman OX40 are regulatory T (Treg) cells.
 138. The method of any one ofclaims 135-137, wherein the depleting is by ADCC and/or phagocytosis.139. The method of any one of claims 134-138, wherein the OX40 agonistantibody binds human OX40 with an affinity of less than or equal toabout 1 nM.
 140. The method of claim 139, wherein the OX40 agonistantibody binds human OX40 with an affinity of less than or equal toabout 0.45 nM.
 141. The method of claim 140, wherein the OX40 agonistantibody binds human OX40 with an affinity of less than or equal toabout 0.4 nM.
 142. The method of any one of claims 134-141, wherein theOX40 agonist antibody binds human OX40 with an EC50 of less than orequal to 0.3 μg/ml.
 143. The method of claim 142, wherein the OX40agonist antibody binds human OX40 with an EC50 of less than or equal to0.2 μg/ml.
 144. The method of any one of claims 134-143, wherein theOX40 agonist antibody increases CD4+ effector T cell proliferationand/or increases cytokine production by the CD4+ effector T cell ascompared to proliferation and/or cytokine production prior to treatmentwith the OX40 agonist antibody.
 145. The method of any one of claims134-144, wherein the OX40 agonist antibody increases memory T cellproliferation and/or cytokine production by a memory T cell.
 146. Themethod of claim 144 or 145, wherein the cytokine production is IFN-γproduction.
 147. The method of any one of claims 134-146, wherein theOX40 agonist antibody inhibits Treg function.
 148. The method of claim147, wherein the OX40 agonist antibody inhibits Treg suppression ofeffector T cell function.
 149. The method of claim 148, wherein effectorT cell function is effector T cell proliferation and/or cytokineproduction.
 150. The method of claim 148 or 149, wherein the effector Tcell is a CD4+ effector T cell.
 151. The method of any one of claims134-150, wherein the OX40 agonist antibody increases OX40 signaltransduction in a target cell that expresses OX40.
 152. The method ofclaim 151, wherein OX40 signal transduction is detected by monitoringNFkB downstream signaling.
 153. The method of any one of claims 134-152,wherein the OX40 agonist antibody comprises a variant IgG1 Fcpolypeptide comprising a mutation that eliminates binding to humaneffector cells and has diminished activity relative to the OX40 agonistantibody comprising a native sequence IgG1 Fc portion.
 154. The methodof claim 153, wherein the OX40 agonist antibody comprises a variant IgG1Fc polypeptide comprising a DANA mutation.
 155. The method of any one ofclaims 134-154, wherein the OX40 agonist antibody comprises (a) a VHdomain comprising (i) a HVR-H1 comprising the amino acid sequence of SEQID NO: 278, 279, or 280, (ii) a HVR-H2 comprising the amino acidsequence of SEQ ID NO: 281, 282, 283, 284, 285, or 286, and (iii) aHVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 287,288, or 289; and (b) a VL domain comprising (i) a HVR-L1 comprising theamino acid sequence of SEQ ID NO: 290, (ii) a HVR-L2 comprising theamino acid sequence of SEQ ID NO: 291, and (iii) a HVR-L3 comprising theamino acid sequence of SEQ ID NO: 292, 293, 294, 295, 296, 297, 298, or299.
 156. The method of claim 155, wherein the OX40 agonist antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:278; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 281;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 287; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 290; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 291; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:
 292. 157. Themethod of claim 155, wherein the OX40 agonist antibody comprises (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 278; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 281; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 287; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 290; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 291; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:
 297. 158. Themethod of claim 155, wherein the OX40 agonist antibody comprises (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 278; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 281; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 287; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 290; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 291; and (f) HVR-L3comprising an amino acid sequence selected from SEQ ID NO:
 298. 159. Themethod of any one of claims 134-158, wherein the OX40 agonist antibodycomprises a VH sequence having at least 90% sequence identity to theamino acid sequence of any one of SEQ ID NOs: 300-325.
 160. The methodof claim 159, wherein the OX40 agonist antibody comprises a VH sequencehaving at least 90% sequence identity to the amino acid sequence of SEQID NO:
 300. 161. The method of claim 160, wherein a total of 1 to 10amino acids have been substituted, inserted, and/or deleted in SEQ IDNO:
 300. 162. The method of any one of claims 159-161, wherein the OX40agonist antibody comprises a VH comprising one, two, or three HVRsselected from: (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 278, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:281, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:287.
 163. The method of any one of claims 134-162, wherein the OX40agonist antibody comprises a VL sequence having at least 90% sequenceidentity to the amino acid sequence of any one of SEQ ID NOs: 326-351.164. The method of claim 163, wherein the OX40 agonist antibodycomprises a VL having at least 90% sequence identity to the amino acidsequence of SEQ ID NO:
 326. 165. The method of claim 164, wherein atotal of 1 to 10 amino acids have been substituted, inserted, and/ordeleted in SEQ ID NO:
 326. 166. The method of any one of claims 162-165,wherein the OX40 agonist antibody comprises a VL comprising one, two, orthree HVRs selected from (a) HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 290; (b) HVR-L2 comprising the amino acid sequence of SEQID NO: 291; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO:
 292. 167. The method of any one of claims 134-166, wherein the OX40agonist antibody comprises (a) a VH sequence of SEQ ID NO: 300; (b) a VLsequence of SEQ ID NO: 326; or (c) a VH sequence as in (a) and a VLsequence as in (b).
 168. The method of any one of claims 134-166,wherein the OX40 agonist antibody comprises (a) a VH sequence of SEQ IDNO: 319; (b) a VL sequence of SEQ ID NO: 345; or (c) a VH sequence as in(a) and a VL sequence as in (b).
 169. The method of any one of claims134-166, wherein the OX40 agonist antibody comprises (a) a VH sequenceof SEQ ID NO: 320; (b) a VL sequence of SEQ ID NO: 346; or (c) a VHsequence as in (a) and a VL sequence as in (b).
 170. The method of anyone of claims 134-154, wherein the OX40 agonist antibody is antibodyL106, antibody ACT35, MEDI6469, or MEDI0562.
 171. The method of any oneof claims 134-170, wherein the OX40 agonist antibody is a full-lengthIgG1 antibody.
 172. The method of claim 134, wherein the OX40immunoadhesin is a trimeric OX40-Fc protein.
 173. The method of any oneof claims 102-172, further comprising administering to the subject anagent that decreases or inhibits one or more additional immuneco-inhibitory receptors.
 174. The method of claim 173, wherein the oneor more additional immune co-inhibitory receptor is selected from thegroup consisting of PD-1, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, andCD96.
 175. The method of any one of claims 102-174, further comprisingadministering to the subject an additional therapeutic agent.
 176. Themethod of claim 175, wherein the additional therapeutic agent is achemotherapeutic agent.
 177. The method of any one of claims 102-176,wherein the antibody is administered subcutaneously, intravenously,intramuscularly, topically, orally, transdermally, intraperitoneally,intraorbitally, by implantation, by inhalation, intrathecally,intraventricularly, or intranasally.
 178. The method of any one ofclaims 102-177, wherein the subject is a human.
 179. A kit comprisingthe antibody of any one of claims 1-70 and a package insert comprisinginstructions for using the antibody for treating or delaying progressionof a cancer in a subject.
 180. A kit comprising the antibody of any oneof claims 1-70 and a package insert comprising instructions for usingthe antibody for treating or delaying progression of an immune-relateddisease in a subject.
 181. A kit comprising the antibody of any one ofclaims 1-70 and a package insert comprising instructions for increasing,enhancing, or stimulating an immune response or function in a subject.182. The kit of any one of claims 179-181, wherein the subject is ahuman.